Structural dynamics of Na+ and Ca2+ interactions with full-size mammalian NCX.

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy.

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

Chloride intracellular channel (CLIC) proteins function as fusogens.

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs' transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

Revealing the tumor suppressive sequence within KL1 domain of the hormone Klotho.

Klotho, a 1012 amino acid transmembrane protein, is a potent tumor suppressor in different cancer types. Klotho is composed of two internal repeats KL1 and KL2, and the tumor suppressor activity is primarily attributed to the KL1 domain. Despite its significant role in regulating various cancer-related pathways, the precise mechanism underlying its tumor suppressor activity remains unresolved. In this study, we aimed to identify the sequence responsible for the tumor suppressor function of Klotho and gain insights into its mechanism of action. To accomplish this, we generated expression vectors of truncated KL1 at the C and N-terminal regions and evaluated their ability to inhibit the colony formation of several cancer cell lines. Our findings demonstrated that truncated KL1 1-340 (KL340) effectively inhibited colony formation similar to KL1, while truncated KL1 1-320 (KL320) lost this activity. Furthermore, this correlated with the inhibitory effect of KL1 and KL340 on the Wnt/ß-catenin pathway, whereas KL320 had no effect. Transcriptomic analysis of MCF-7 cells expressing the constructs revealed enriched pathways associated with tumor suppressor activity in KL1 and KL340. Interestingly, the α-fold predictor tool highlighted distinct differences in the α and ß sheets of the TIM barrel fold of the truncated Klotho constructs, adding to our understanding of their structural variations. In summary, this study identified the 340 N-terminal amino acids as the sequence that possesses Klotho's tumor suppressor activity and reveals a critical role in the 320-340 sequence for this function. It also provides a foundation for the development of Klotho-based therapeutic approaches for cancer treatment.

Tumor Treating Fields (TTFields) demonstrate antiviral functions in vitro, and safety for application to COVID-19 patients in a pilot clinical study.

Coronaviruses are the causative agents of several recent outbreaks, including the COVID-19 pandemic. One therapeutic approach is blocking viral binding to the host receptor. As binding largely depends on electrostatic interactions, we hypothesized possible inhibition of viral infection through application of electric fields, and tested the effectiveness of Tumor Treating Fields (TTFields), a clinically approved cancer treatment based on delivery of electric fields. In preclinical models, TTFields were found to inhibit coronavirus infection and replication, leading to lower viral secretion and higher cell survival, and to formation of progeny virions with lower infectivity, overall demonstrating antiviral activity. In a pilot clinical study (NCT04953234), TTFields therapy was safe for patients with severe COVID-19, also demonstrating preliminary effectiveness data, that correlated with higher device usage.

Tumor Treating Fields (TTFields) increase the effectiveness of temozolomide and lomustine in glioblastoma cell lines.

PURPOSE: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. TTFields therapy is approved for treatment of newly-diagnosed glioblastoma (GBM) concurrent with maintenance temozolomide (TMZ). Recently, the benefit of TMZ in combination with lomustine (CCNU) was demonstrated in patients with O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. The addition of adjuvant TTFields to TMZ plus CCNU further improved patient outcomes, leading to a CE mark for this regimen. The current in vitro study aimed to elucidate the mechanism underlying the benefit of this treatment protocol. METHODS: Human GBM cell lines with different MGMT promoter methylation statuses were treated with TTFields, TMZ, and CCNU, and effectiveness was tested by cell count, apoptosis, colony formation, and DNA damage measurements. Expression levels of relevant DNA-repair proteins were examined by western blot analysis. RESULTS: TTFields concomitant with TMZ displayed an additive effect, irrespective of MGMT expression levels. TTFields concomitant with CCNU or with CCNU plus TMZ was additive in MGMT-expressing cells and synergistic in MGMT-non-expressing cells. TTFields downregulated the FA-BRCA pathway and increased DNA damage induced by the chemotherapy combination. CONCLUSIONS: The results support the clinical benefit demonstrated for TTFields concomitant with TMZ plus CCNU. Since the FA-BRCA pathway is required for repair of DNA cross-links induced by CCNU in the absence of MGMT, the synergy demonstrated in MGMT promoter methylated cells when TTFields and CCNU were co-applied may be attributed to the BRCAness state induced by TTFields.

Defective cathepsin Z affects EGFR expression and causes autosomal dominant palmoplantar keratoderma.

BACKGROUND: The abnormal function of epidermal growth factor receptor (EGFR) has recently been shown to underlie various disorders of cornification. OBJECTIVES: To delineate the genetic basis of a novel dominant form of palmoplantar keratoderma (PPK). METHODS: Whole-exome (WES) and direct sequencing, quantitative real-time polymerase chain reaction, protein modelling, confocal immunofluorescence microscopy, immunoblotting, three-dimensional skin equivalents and an enzyme activity assay were used to delineate the genetic basis of a novel dominant form of PPK. RESULTS: WES revealed heterozygous variants (c.274T > C and c.305C > T) in CTSZ (encoding cathepsin Z) in four individuals (belonging to three unrelated families) with focal PPK. Bioinformatics and protein modelling predicted the variants to be pathogenic. Previous studies have suggested that EGFR expression may be subject to cathepsin regulation. Immunofluorescence revealed reduced cathepsin Z expression in the upper epidermal layers and concomitant increased epidermal EGFR expression in patients harbouring CTSZ variants. Accordingly, human keratinocytes transfected with constructs expressing PPK-causing variants in CTSZ displayed reduced cathepsin Z enzymatic activity, as well as increased EGFR expression. In line with the role played by EGFR in the regulation of keratinocyte proliferation, human keratinocytes transfected with the PPK-causing variants showed significantly increased proliferation that was abolished upon exposure to erlotinib, an EGFR inhibitor. Similarly, downregulation of CTSZ resulted in increased EGFR expression and increased proliferation in human keratinocytes, suggestive of a loss-of-function effect of the pathogenic variants. Finally, three-dimensional organotypic skin equivalents grown from CTSZ-downregulated cells showed increased epidermal thickness and EGFR expression as seen in patient skin; here, too, erlotinib was found to rescue the abnormal phenotype. CONCLUSIONS: Taken collectively, these observations attribute to cathepsin Z a hitherto unrecognized function in epidermal differentiation.

Aurora B Kinase Inhibition by AZD1152 Concomitant with Tumor Treating Fields Is Effective in the Treatment of Cultures from Primary and Recurrent Glioblastomas.

Tumor Treating Fields (TTFields) were incorporated into the treatment of glioblastoma, the most malignant brain tumor, after showing an effect on progression-free and overall survival in a phase III clinical trial. The combination of TTFields and an antimitotic drug might further improve this approach. Here, we tested the combination of TTFields with AZD1152, an Aurora B kinase inhibitor, in primary cultures of newly diagnosed (ndGBM) and recurrent glioblastoma (rGBM). AZD1152 concentration was titrated for each cell line and 5-30 nM were used alone or in addition to TTFields (1.6 V/cm RMS; 200 kHz) applied for 72 h using the inovitro™ system. Cell morphological changes were visualized by conventional and confocal laser microscopy. The cytotoxic effects were determined by cell viability assays. Primary cultures of ndGBM and rGBM varied in p53 mutational status; ploidy; EGFR expression and MGMT-promoter methylation status. Nevertheless; in all primary cultures; a significant cytotoxic effect was found following TTFields treatment alone and in all but one, a significant effect after treatment with AZD1152 alone was also observed. Moreover, in all primary cultures the combined treatment had the most pronounced cytotoxic effect in parallel with morphological changes. The combined treatment of TTFields and AZD1152 led to a significant reduction in the number of ndGBM and rGBM cells compared to each treatment alone. Further evaluation of this approach, which has to be considered as a proof of concept, is warranted, before entering into early clinical trials.

Correction: Davidi et al. Tumor Treating Fields (TTFields) Concomitant with Sorafenib Inhibit Hepatocellular Carcinoma In Vitro and In Vivo. Cancers 2022, 14, 2959.

The authors wish to make minor corrections to Figure 1 and Figure 2 of the following paper [...].

Tumor Treating Fields (TTFields) Concomitant with Immune Checkpoint Inhibitors Are Therapeutically Effective in Non-Small Cell Lung Cancer (NSCLC) In Vivo Model.

Tumor Treating Fields (TTFields) are electric fields that exert physical forces to disrupt cellular processes critical for cancer cell viability and tumor progression. TTFields induce anti-mitotic effects through the disruption of the mitotic spindle and abnormal chromosome segregation, which trigger several forms of cell death, including immunogenic cell death (ICD). The efficacy of TTFields concomitant with anti-programmed death-1 (anti-PD-1) treatment was previously shown in vivo and is currently under clinical investigation. Here, the potential of TTFields concomitant with anti- PD-1/anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed death-ligand 1 (anti-PD-L1) immune checkpoint inhibitors (ICI) to improve therapeutic efficacy was examined in lung tumor-bearing mice. Increased circulating levels of high mobility group box 1 protein (HMGB1) and elevated intratumoral levels of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α) were found in the TTFields-treated mice, indicative of ICD induction. The concomitant application of TTFields and ICI led to a significant decrease in tumor volume as compared to all other groups. In addition, significant increases in the number of tumor-infiltrating immune cells, specifically cytotoxic T-cells, were observed in the TTFields plus anti-PD-1/anti-CTLA-4 or anti-PD-L1 groups. Correspondingly, cytotoxic T-cells isolated from these tumors showed higher levels of IFN-γ production. Collectively, these results suggest that TTFields have an immunoactivating role that may be leveraged for concomitant treatment with ICI to achieve better tumor control by enhancing antitumor immunity.

Tumor Treating Fields (TTFields) Reversibly Permeabilize the Blood-Brain Barrier In Vitro and In Vivo.

Despite the availability of numerous therapeutic substances that could potentially target CNS disorders, an inability of these agents to cross the restrictive blood-brain barrier (BBB) limits their clinical utility. Novel strategies to overcome the BBB are therefore needed to improve drug delivery. We report, for the first time, how Tumor Treating Fields (TTFields), approved for glioblastoma (GBM), affect the BBB's integrity and permeability. Here, we treated murine microvascular cerebellar endothelial cells (cerebEND) with 100-300 kHz TTFields for up to 72 h and analyzed the expression of barrier proteins by immunofluorescence staining and Western blot. In vivo, compounds normally unable to cross the BBB were traced in healthy rat brain following TTFields administration at 100 kHz. The effects were analyzed via MRI and immunohistochemical staining of tight-junction proteins. Furthermore, GBM tumor-bearing rats were treated with paclitaxel (PTX), a chemotherapeutic normally restricted by the BBB combined with TTFields at 100 kHz. The tumor volume was reduced with TTFields plus PTX, relative to either treatment alone. In vitro, we demonstrate that TTFields transiently disrupted BBB function at 100 kHz through a Rho kinase-mediated tight junction claudin-5 phosphorylation pathway. Altogether, if translated into clinical use, TTFields could represent a novel CNS drug delivery strategy.

Varicocoele in adolescence and testicular cancer in young adulthood.

BACKGROUND: Elevated intrascrotal temperature has been suggested as a risk factor for testicular cancer, which is the most common neoplasm among young men. Varicocoele was linked to increased intrascrotal temperature, but whether it is associated with testicular cancer is unclear. OBJECTIVE: To explore the possible association between varicocoele at adolescence and the incidence of testicular cancer at adulthood. DESIGN, SETTING, AND PARTICIPANTS: This nationwide, population-based, historical cohort study includes 1,521,661 Israeli male adolescents (mean age 17.5 ± 0.4 years), who were screened for varicocoele during the years 1967-2012, as part of their medical assessment prior to compulsory military service. The mean follow-up was 18 ± 4.2 years. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The diagnosis of testicular cancer was ascertained from linkage of records to the the Israeli National Cancer Registry. Survival analysis was applied. RESULTS: In total, 53,210 adolescents were diagnosed with varicocoele stages 2 and 3 prior to military service. Of 1988 (0.13% of the total cohort) men who were diagnosed with testicular cancer during follow-up, 54 (0.1%) had varicocoele prior to military service, while 1934 (99.9%) did not; p = 0.213. The age at cancer diagnosis and the distribution of seminomas versus non-seminomas did not differ significantly between those with and without varicocoele in adolescence. In a multivariable analysis controlling for sociodemographic factors, varicocoele was not associated with testicular cancer; odds ratio = 0.816 (CI: 0.615-1.083). CONCLUSIONS: Varicocoele in adolescents was not found to be associated with testicular cancer in young adults. PATIENT SUMMARY: In light of the theoretical association between varicocoele and testicular cancer, we conducted this large population study. We found no association between varicocoele in young adulthood and testicular cancer later in life.

Exploring the Li+ transporting mutant of NCX_Mj for assigning ion binding sites of mitochondrial NCLX.

The plasma membrane (NCX) and mitochondrial (NCLX) Na+/Ca2+ exchangers are structurally related proteins, although they operate under strictly different ionic conditions and membrane potentials. In contrast with NCX, NCLX can transport either Li+ or Na+ in exchange for Ca2+. Whereas the crystal structure of the archaeal NCX (NCX_Mj) describes the binding sites for alternative binding of 3Na+ or 1Ca2+, these features remain elusive for NCLX due to the lack of structural information. To elucidate the ion-binding features of mitochondrial NCLX, we analyzed here the Li+-transporting NCLX_Mj mutant, produced by replacing the ion-coordinating residues in the archaeal NCX (NCX_Mj) to match the ion-coordinating residues of human NCLX. The NCLX_Mj-mediated Na+/Ca2+ or Li+/Ca2+ exchange rates are insensitive to varying voltage, consistent with an electroneutral ion exchange. Molecular dynamics (MD) simulations revealed that NCLX_Mj contains two novel Li+ binding sites with four ion-coordinating residues, derived from the three Na+ binding sites of NCX_Mj. The ion-coordination modes, observed in the MD simulations, were further supported by two-dimensional infrared (2D IR) spectroscopy and by testing the mutational effects on the ion fluxes. Collectively, our results revealed a structural basis for Li+ binding and electroneutral transport (2Na+/Li+:1Ca2+) by NCLX_Mj, meaning that the NCLX-mediated electroneutral transport may predefine mitochondrial Ca2+ and Na+ signaling to modulate cellular functions.

TTYH family members form tetrameric complexes at the cell membrane.

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs shared a dimeric stoichiometry following detergent solubilization, their structures revealed divergence in their relative subunit orientation. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrate that mTTYH1 and mTTYH3 form tetramers at the plasma membrane, stabilized by interactions between their extracellular domains. Using blue-native PAGE, fluorescence-detection size-exclusion chromatography, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we reveal that detergent solubilization results in tetramers destabilization, leading to their dissolution into dimers. Moreover, HDX-MS demonstrates that the extracellular domains are stabilized in the context of the tetrameric mTTYH complex. Together, our results expose the innate tetrameric organization of TTYH complexes at the cell membrane. Future structural analyses of these assemblies in native membranes are required to illuminate their long-sought cellular function.

Tumor Treating Fields (TTFields) Concomitant with Sorafenib Inhibit Hepatocellular Carcinoma In Vitro and In Vivo.

Hepatocellular carcinoma (HCC), a highly aggressive liver cancer, is a leading cause of cancer-related death. Tumor Treating Fields (TTFields) are electric fields that exert antimitotic effects on cancerous cells. The aims of the current research were to test the efficacy of TTFields in HCC, explore the underlying mechanisms, and investigate the possible combination of TTFields with sorafenib, one of the few front-line treatments for patients with advanced HCC. HepG2 and Huh-7D12 human HCC cell lines were treated with TTFields at various frequencies to determine the optimal frequency eliciting maximal cell count reduction. Clonogenic, apoptotic effects, and autophagy induction were measured. The efficacy of TTFields alone and with concomitant sorafenib was tested in cell cultures and in an orthotopic N1S1 rat model. Tumor volume was examined at the beginning and following 5 days of treatment. At study cessation, tumors were weighed and examined by immunohistochemistry to assess autophagy and apoptosis. TTFields were found in vitro to exert maximal effect at 150 kHz, reducing cell count and colony formation, increasing apoptosis and autophagy, and augmenting the effects of sorafenib. In animals, TTFields concomitant with sorafenib reduced tumor weight and volume fold change, and increased cases of stable disease following treatment versus TTFields or sorafenib alone. While each treatment alone elevated levels of autophagy relative to control, TTFields concomitant with sorafenib induced a significant increase versus control in tumor ER stress and apoptosis levels, demonstrating increased stress under the multimodal treatment. Overall, TTFields treatment demonstrated efficacy and enhanced the effects of sorafenib for the treatment of HCC in vitro and in vivo, via a mechanism involving induction of autophagy.

Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases.

Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

Tumor Treating Fields (TTFields) downregulate the Fanconi Anemia-BRCA pathway and increase the efficacy of chemotherapy in malignant pleural mesothelioma preclinical models.

OBJECTIVES: Tumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields with antimitotic effects on cancerous cells. TTFields concomitant with pemetrexed and a platinum agent are approved in the US and EU as first line therapy for unresectable, locally advanced or metastatic malignant pleural mesothelioma (MPM). The goal of the current study was to characterize the mechanism of action of TTFields in MPM cell lines and animal models. METHODS: Human MPM cell lines MSTO-211H and NCI-H2052 were treated with TTFields to determine the frequency that elicits maximal cytotoxicity. The effect of TTFields on DNA damage and repair, and the cytotoxic effect of TTFields in combination with cisplatin and/or pemetrexed were examined. Efficacy of TTFields concomitant with cisplatin and pemetrexed was evaluated in orthotopic IL-45 and subcutaneous RN5 murine models. RESULTS: TTFields at a frequency of 150 kHz demonstrated the highest cytotoxicity to MPM cells. Application of 150 kHz TTFields resulted in increased formation of DNA double strand breaks, elevated expression of DNA damage induced cell cycle arrest proteins, and reduced expression of Fanconi Anemia (FA)-BRCA DNA repair pathway proteins. Co-treatment of TTFields with cisplatin or pemetrexed significantly increased treatment efficacy versus each modality alone, with additivity and synergy exhibited by the TTFields-pemetrexed and TTFields-cisplatin combinations, respectively. In animal models, tumor volume was significantly lower for the TTFields-cisplatin-pemetrexed combination compared to control, accompanied by increased DNA damage within the tumor. CONCLUSION: This research demonstrated that the efficacy of TTFields for the treatment of MPM is associated with reduced expression of FA-BRCA pathway proteins and increased DNA damage. This mechanism of action is consistent with the observed synergism for TTFields-cisplatin vs additivity for TTFields-pemetrexed, as cisplatin-induced DNA damage is repaired via the FA-BRCA pathway.