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.

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.

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.

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.

Conserved cysteine dioxidation enhances membrane interaction of human Cl- intracellular channel 5.

The human chloride intracellular channel (hCLIC) family is thought to transition between globular and membrane-associated forms by exposure of a hydrophobic surface. However, the molecular identity of this surface, and the triggering events leading to its exposure, remain elusive. Here, by combining biochemical and structural approaches, together with mass spectrometry (MS) analyses, we show that hCLIC5 is inherently flexible. X-ray crystallography revealed the existence of a globular conformation, while small-angle X-ray scattering showed additional elongated forms consisting of exposure of the conserved hydrophobic inter-domain interface to the bulk phase. Tryptophan fluorescence measurements demonstrated that the transition to the membrane-associated form is enhanced by the presence of oxidative environment and lipids. Using MS, we identified a dose-dependent oxidation of a highly conserved cysteine residue, known to play a key role in the structurally related omega-class of glutathione-S-transferases. Hydrogen/deuterium exchange MS analysis revealed that oxidation of this cysteine facilitates the exposure of the conserved hydrophobic inter-domain interface. Together, our results pinpoint an oxidation of a specific cysteine residue as a triggering mechanism initializing the molecular commitment for membrane interaction in the CLIC family.

Tumor-treating fields (TTFields) induce immunogenic cell death resulting in enhanced antitumor efficacy when combined with anti-PD-1 therapy.

Tumor-treating fields (TTFields) are alternating electric fields in a specific frequency range (100-300 kHz) delivered to the human body through transducer arrays. In this study, we evaluated whether TTFields-mediated cell death can elicit antitumoral immunity and hence would be effectively combined with anti-PD-1 therapy. We demonstrate that in TTFields-treated cancer cells, damage-associated molecular patterns including high-mobility group B1 and adenosine triphosphate are released and calreticulin is exposed on the cell surface. Moreover, we show that TTFields treatment promotes the engulfment of cancer cells by dendritic cells (DCs) and DCs maturation in vitro, as well as recruitment of immune cells in vivo. Additionally, our study demonstrates that the combination of TTFields with anti-PD-1 therapy results in a significant decline of tumor volume and increase in the percentage of tumor-infiltrating leukocytes in two tumor models. In orthotopic lung tumors, these infiltrating leukocytes, specifically macrophages and DCs, showed elevated expression of PD-L1. Compatibly, cytotoxic T-cells isolated from these tumors demonstrated increased production of IFN-γ. In colon cancer tumors, T-cells infiltration was significantly increased following long treatment duration with TTFields plus anti-PD-1. Collectively, our results suggest that TTFields therapy can induce anticancer immune response. Furthermore, we demonstrate robust efficacy of concomitant application of TTFields and anti-PD-1 therapy. These data suggest that integrating TTFields with anti-PD-1 therapy may further enhance antitumor immunity, hence achieve better tumor control.

Convulsive seizures and some behavioral comorbidities are uncoupled in the Scn1aA1783V Dravet syndrome mouse model.

OBJECTIVE: Dravet syndrome (Dravet) is a severe childhood epileptic encephalopathy. The disease begins with a febrile stage, characterized by febrile seizures with otherwise normal development. Progression to the worsening stage features recurrent intractable seizures and the presentation of additional nonepileptic comorbidities, including global developmental delay, hyperactivity, and motor deficits. Later in life, at the stabilization stage, seizure burden decreases, whereas Dravet-associated comorbidities persist. To date, it remains debated whether the nonepileptic comorbidities result from severe epilepsy or represent an independent phenotypic feature. METHODS: Dravet mice (DS) faithfully recapitulate many clinical aspects of Dravet. Using wild-type (WT) and DS at different ages, we monitored multiple behavioral features as well as background electroencephalogram (EEG) activity during the different stages of Dravet epilepsy. RESULTS: Behavioral tests of WT and DS demonstrated that some deficits manifest already at the pre-epileptic stage, prior to the onset of convulsive seizures. These include motor impairment and hyperactivity in the open field. Deficits in cognitive functions were detected at the onset of severe spontaneous seizures. Power spectral analysis of background EEG activity, measured through development, showed that DS exhibit normal background oscillations at the pre-epileptic stage, a marked reduction in total power during the onset of severe epilepsy, and a subsequent smaller reduction later in life. Importantly, low EEG power at the stage of severe frequent convulsive seizures correlated with increased risk for premature death. SIGNIFICANCE: Our data provide a comprehensive developmental trajectory of Dravet epilepsy and Dravet-associated comorbidities in mice, under controlled settings, demonstrating that the convulsive seizures and some nonepileptic comorbidities may be uncoupled. Moreover, we report the existence of an inverse correlation, on average, between the power of background EEG and the severity of epileptic phenotypes, suggesting that such measurements may potentially serve as a biomarker for Dravet severity.

Metal Coordination Is Crucial for Geranylgeranyl Diphosphate Synthase-Bisphosphonate Interactions: A Crystallographic and Computational Analysis.

Geranylgeranyl diphosphate synthase (GGPPS) is a central metalloenzyme in the mevalonate pathway, crucial for the prenylation of small GTPases. As small GTPases are pivotal for cellular survival, GGPPS was highlighted as a potential target for treating human diseases, including solid and hematologic malignancies and parasitic infections. Most available GGPPS inhibitors are bisphosphonates, but the clinically available compounds demonstrate poor pharmacokinetic properties. Although the design of novel bisphosphonates with improved physicochemical properties is highly desirable, the structure of wild-type human GGPPS (hGGPPS) bound to a bisphosphonate has not been resolved. Moreover, various metal-bisphosphonate-binding stoichiometries were previously reported in structures of yeast GGPPS (yGGPPS), hampering computational drug design with metal-binding pharmacophores (MBP). In this study, we report the 2.2 Å crystal structure of hGGPPS in complex with ibandronate, clearly depicting the involvement of three Mg2+ ions in bisphosphonate-protein interactions. Using drug-binding assays and computational docking, we show that the assignment of three Mg2+ ions to the binding site of both hGGPPS and yGGPPS greatly improves the correlation between calculated binding energies and experimentally measured affinities. This work provides a structural basis for future rational design of additional MBP-harboring drugs targeting hGGPPS. SIGNIFICANCE STATEMENT: Bisphosphonates are inhibitors of geranylgeranyl diphosphate synthase (GGPPS), a metalloenzyme crucial for cell survival. Bisphosphonate binding depends on coordination by Mg2+ ions, but various Mg2+-bisphosphonate-binding stoichiometries were previously reported. In this study, we show that three Mg2+ ions are vital for drug binding and provide a structural basis for future computational design of GGPPS inhibitors.

Reduced Activity of Geranylgeranyl Diphosphate Synthase Mutant Is Involved in Bisphosphonate-Induced Atypical Fractures.

Bisphosphonates are widely used for treating osteoporosis, a common disorder in which bone strength is reduced, increasing the risk for fractures. Rarely, bisphosphonates can paradoxically lead to atypical fractures occurring spontaneously or with trivial trauma. Recently, a novel missense mutation (D188Y) in the GGPS1 gene, encoding for geranylgeranyl diphosphate synthase (GGPPS), was associated with bisphosphonate-induced atypical fractures. However, the molecular basis for GGPPS involvement in this devastating condition remains elusive. Here, we show that while maintaining an overall unperturbed global enzyme structure, the D188Y mutation leads to ∼4-fold catalytic activity decrease. Furthermore, GGPPS-D188Y is unable to support cross-species complementation, highlighting the functional significance of the reduced catalytic activity observed in vitro. We next determined the crystal structure of apo-GGPPS-D188Y, revealing that while Y188 does not alter the protein fold, its bulky side chain sterically interferes with substrate binding. In agreement, we show that GGPPS-D188Y exhibits ∼3-fold reduction in the binding affinity of zoledronate, a commonly used bisphosphonate. However, inhibition of the mutated enzyme by zoledronate, in pharmacologically relevant concentrations, is maintained. Finally, we determined the crystal structure of zoledronate-bound GGPPS-D188Y, revealing large ligand-induced binding pocket rearrangements, revising the previous model for GGPPS-bisphosphonate interactions. In conclusion, we propose that among heterozygotes residual GGPPS activity is sufficient to support physiologic cellular function, concealing any pathologic phenotype. However, under bisphosphonate treatment, GGPPS activity is reduced below a crucial threshold for osteoclast function, leading to impaired bone remodeling and increased susceptibility to atypical fractures.

Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states.

Na+/Ca2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na+ or Ca2+ specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

Structure-dynamic basis of splicing-dependent regulation in tissue-specific variants of the sodium-calcium exchanger.

Tissue-specific splice variants of Na(+)/Ca(2+) exchangers contain 2 Ca(2+)-binding regulatory domains (CBDs), CBD1 and CBD2. Ca(2+) interaction with CBD1 activates sodium-calcium exchangers (NCXs), and Ca(2+) binding to CBD2 alleviates Na(+)-dependent inactivation. A combination of mutually exclusive (A, B) and cassette (C-F) exons in CBD2 raises functionally diverse splice variants through unknown mechanisms. Here, the effect of exons on CBDs backbone dynamics were investigated in the 2-domain tandem (CBD12) of the brain, kidney, and cardiac splice variants by using hydrogen-deuterium exchange mass spectrometry and stopped-flow techniques. Mutually exclusive exons stabilize interdomain interactions in the apoprotein, which primarily predefines the extent of responses to Ca(2+) binding. Deuterium uptake levels were up to 20% lower in the cardiac vs. the brain CBD12, reveling that elongation of the CBD2 FG loop by cassette exons rigidifies the interdomain Ca(2+) salt bridge at the 2-domain interface, which secondarily modulates the Ca(2+)-bound states. In matching splice variants, the extent of Ca(2+)-induced rigidification correlates with decreased (up to 10-fold) Ca(2+) off rates, where the cardiac CBD12 exhibits the slowest Ca(2+) off rates. Collectively, structurally disordered/dynamic segments at mutually exclusive and cassette exons have local and distant effects on the folded structures nearby the Ca(2+) binding sites, which may serve as a structure-dynamic basis for splicing-dependent regulation of NCX.

Purification and characterization of human dehydrodolychil diphosphate synthase (DHDDS) overexpressed in E. coli.

Protein asparagine (N)-linked glycosylation is a post-translational modification that occurs in the endoplasmic reticulum; it plays an important role in protein folding, oligomerization, quality control, sorting, and transport. Accordingly, disorders of glycosylation may affect practically every organ system. Dehydrodolichyl diphosphate synthase (DHDDS) is an eukaryotic cis prenyltransferase (cis-PT) that catalyzes chain elongation of farnesyl diphosphate via multiple condensations with isopentenyl diphosphate to form dehydrodolichyl diphosphate, a precursor for the glycosyl carrier dolichylpyrophophate involved in N-linked glycosylation. Mutations in DHDDS were shown to result in retinitis pigmentosa, ultimately leading to blindness, but the exact molecular mechanism by which the mutations affect DHDDS function remains elusive. In addition, bacterial cis-PT homologs are involved in bacterial wall synthesis and are therefore potential targets for new antibacterial agents. However, as eukaryotic cis-PT were not thoroughly characterized structurally and functionally, rational design of prokaryotic cis-PT specific drugs is currently impossible. Here, we present a simple protocol for purification of functionally active human DHDDS under non-denaturating conditions using a codon-optimized construct. The purified protein forms a stable homodimer, similar to its bacterial homologs, and shows time- and substrate-dependent activity. Purification of this protein requires the presence of a detergent for protein solubility. The protocol described here may be utilized for the overexpression of other eukaryotic cis-PT. Future structural and functional studies of the recombinant DHDDS may shed light on the mechanisms underlying DHDDS-related retinitis pigmentosa and lead to novel therapeutic approaches.

Perioperative complications of emergent and elective procedures in psychiatric patients.

BACKGROUND: Patients with psychiatric disorders have an increased risk for morbidity and mortality from other medical conditions. METHODS: Medical records of all the patients undergoing appendectomy (n = 2594) and laparoscopic cholecystectomy (n = 2874) from 2009 to 2014 in one hospital were reviewed. For each patient with a documented psychiatric disorder undergoing surgery, four controls were matched. RESULTS: The final sample of patients undergoing appendectomy included 96 patients, whereas those undergoing laparoscopic cholecystectomy included 260 patients. In the emergent scenario, psychiatric patients had longer time from symptom appearance to admission, longer hospitalization duration, and increased rate and severity of postoperative complications. In the elective scenario, psychiatric patients were shown to have more postoperative respiratory complications. CONCLUSIONS: Our results, together with the high prevalence of psychiatric disorders in the population, underscore the importance of screening for psychiatric disorders and their proper documentation in surgical patients.

Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation.

Na(+)/Ca(2+) exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca(2+) homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na(+) or Ca(2+), and thus can catalyze uphill Ca(2+) transport driven by a Na(+) gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na(+) ions occupy the so-called Sext, SCa, and Sint sites, whereas the Smid site is occupied by one water molecule and one H(+), which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na(+)/Ca(2+) and Ca(2+)/Ca(2+) exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na(+) and Ca(2+) requires protonation of D240, and that this side chain does not coordinate either ion at Smid. These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na(+) across the membrane is electrogenic, whereas transport of Ca(2+) is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.

Structure-Based Engineering of Lithium-Transport Capacity in an Archaeal Sodium-Calcium Exchanger.

Members of the Ca(2+)/cation exchanger superfamily (Ca(2+)/CA) share structural similarities (including highly conserved ion-coordinating residues) while exhibiting differential selectivity for Ca(2+), Na(+), H(+), K(+), and Li(+). The archaeal Na(+)/Ca(2+) exchanger (NCX_Mj) and its mammalian orthologs are highly selective for Na(+), whereas the mitochondrial ortholog (NCLX) can transport either Li(+) or Na(+) in exchange with Ca(2+). Here, structure-based replacement of ion-coordinating residues in NCX_Mj resulted in a capacity for transporting either Na(+) or Li(+), similar to the case for NCLX. This engineered protein may serve as a model for elucidating the mechanisms underlying ion selectivity and ion-coupled alternating access in NCX and similar proteins.

Alternating electric fields (TTFields) in combination with paclitaxel are therapeutically effective against ovarian cancer cells in vitro and in vivo.

Long-term survival rates for advanced ovarian cancer patients have not changed appreciably over the past four decades; therefore, development of new, effective treatment modalities remains a high priority. Tumor Treating Fields (TTFields), a clinically active anticancer modality utilize low-intensity, intermediate frequency, alternating electric fields. The goal of this study was to evaluate the efficacy of combining TTFields with paclitaxel against ovarian cancer cells in vitro and in vivo. In vitro application of TTFields on human ovarian cancer cell lines led to a significant reduction in cell counts as compared to untreated cells. The effect was found to be frequency and intensity dependent. Further reduction in the number of viable cells was achieved when TTFields treatment was combined with paclitaxel. The in vivo effect of the combined treatment was tested in mice orthotopically implanted with MOSE-LTICv cells. In this model, combined treatment led to a significant reduction in tumor luminescence and in tumor weight as compared to untreated mice. The feasibility of effective local delivery of TTFields to the human abdomen was examined using finite element mesh simulations performed using the Sim4life software. These simulations demonstrated that electric fields intensities inside and in the vicinity of the ovaries of a realistic human computational phantom are about 1 and 2 V/cm pk-pk, respectively, which is within the range of intensities required for TTFields effect. These results suggest that prospective clinical investigation of the combination of TTFields and paclitaxel is warranted.

Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins.

The Ca(2+)-dependent allosteric regulation of Na(+)/Ca(2+) exchanger (NCX) proteins represents Ca(2+) interaction with the cytosolic domains, CBD1 (calcium-binding domain 1) and CBD2, which is associated either with activation, inhibition or no response to regulatory Ca(2+) in a given splice variant. CBD1 contains a high affinity Ca(2+)-sensor (which is highly conserved among splice variants), whereas primary information upon Ca(2+) binding to CBD1 is modified by alternative splicing of CBD2, yielding the diverse regulatory responses to Ca(2+). To resolve the structure-dynamic determinants of splicing-dependent regulation, we tested two-domain tandem (CBD12) constructs possessing either positive, negative or no response to Ca(2+) using hydrogen-deuterium exchange MS (HDX-MS), SAXS, equilibrium 45Ca(2+) binding and stopped-flow kinetics. Taken together with previously resolved crystallographic structures of CBD12, the data revealed that Ca(2+) binding to CBD1 rigidifies the main-chain flexibility of CBD2 (but not of CBD1), whereas CBD2 stabilizes the apo-CBD1. Strikingly, the extent and strength of Ca(2+)-dependent rigidification of CBD2 is splice-variant dependent, where the main-chain rigidification spans from the Ca(2+)-binding sites of CBD1, through a helix of CBD2 (positioned at the domains' interface) up to the tip of CBD2 [>50 Å (1 Å = 0.1 nm)] or alternatively, it stops at the CBD2 helix in the splice variant exhibiting an inhibitory response to regulatory Ca(2+). These results provide a structure-dynamic basis by which alternative splicing diversifies the regulatory responses to Ca(2+) as well as controls the extent and strength of allosteric signal propagation over long distance.

Structure-Functional Basis of Ion Transport in Sodium-Calcium Exchanger (NCX) Proteins.

The membrane-bound sodium-calcium exchanger (NCX) proteins shape Ca2+ homeostasis in many cell types, thus participating in a wide range of physiological and pathological processes. Determination of the crystal structure of an archaeal NCX (NCX_Mj) paved the way for a thorough and systematic investigation of ion transport mechanisms in NCX proteins. Here, we review the data gathered from the X-ray crystallography, molecular dynamics simulations, hydrogen-deuterium exchange mass-spectrometry (HDX-MS), and ion-flux analyses of mutants. Strikingly, the apo NCX_Mj protein exhibits characteristic patterns in the local backbone dynamics at particular helix segments, thereby possessing characteristic HDX profiles, suggesting structure-dynamic preorganization (geometric arrangements of catalytic residues before the transition state) of conserved α1 and α2 repeats at ion-coordinating residues involved in transport activities. Moreover, dynamic preorganization of local structural entities in the apo protein predefines the status of ion-occlusion and transition states, even though Na⁺ or Ca2+ binding modifies the preceding backbone dynamics nearby functionally important residues. Future challenges include resolving the structural-dynamic determinants governing the ion selectivity, functional asymmetry and ion-induced alternating access. Taking into account the structural similarities of NCX_Mj with the other proteins belonging to the Ca2+/cation exchanger superfamily, the recent findings can significantly improve our understanding of ion transport mechanisms in NCX and similar proteins.

Population shift underlies Ca2+-induced regulatory transitions in the sodium-calcium exchanger (NCX).

In eukaryotic Na(+)/Ca(2+) exchangers (NCX) the Ca(2+) binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca(2+) sensor (Ca3-Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca(2+)-driven interdomain switch has been described, albeit how it couples Ca(2+) binding with signal propagation remains unclear. To resolve the dynamic features of Ca(2+)-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium (45)Ca(2+) binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg(2+)-bound forms of CBD12 are highly flexible, whereas Ca(2+) binding to the Ca3-Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca(2+) is observed on the globally derived Dmax (maximal interatomic distance), although under comparable conditions a normal [Ca(2+)]-dependent allosteric regulation occurs. Low affinity sites (Ca1-Ca2) of CBD1 do not contribute to Ca(2+)-induced population shift, but the occupancy of these sites by 1 mM Mg(2+) shifts the Ca(2+) affinity (Kd) at the neighboring Ca3-Ca4 sites from ∼ 50 nM to ∼ 200 nM and thus, keeps the primary Ca(2+) sensor (Ca3-Ca4 sites) within a physiological range. Thus, Ca(2+) binding to the Ca3-Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.