Structural basis of autoinhibition of the human NHE3-CHP1 complex.

Sodium-proton exchanger 3 (NHE3/SLC9A3) located in the apical membrane of renal and gastrointestinal epithelia mediates salt and fluid absorption and regulates pH homeostasis. As an auxiliary regulatory factor of NHE proteins, calcineurin B homologous protein 1 (CHP1) facilitates NHE3 maturation, plasmalemmal expression, and pH sensitivity. Dysfunctions of NHE3 are associated with renal and digestive system disorders. Here, we report the cryo-electron microscopy structure of the human NHE3-CHP1 complex in its inward-facing conformation. We found that a cytosolic helix-loop-helix motif in NHE3 blocks the intracellular cavity formed between the core and dimerization domains, functioning as an autoinhibitory element and hindering substrate transport. Furthermore, two phosphatidylinositol molecules are found to bind to the peripheric juxtamembrane sides of the complex, function as anchors to stabilize the complex, and may thus enhance its transport activity.

Structure and mechanism of the human NHE1-CHP1 complex.

Sodium/proton exchanger 1 (NHE1) is an electroneutral secondary active transporter present on the plasma membrane of most mammalian cells and plays critical roles in regulating intracellular pH and volume homeostasis. Calcineurin B-homologous protein 1 (CHP1) is an obligate binding partner that promotes NHE1 biosynthetic maturation, cell surface expression and pH-sensitivity. Dysfunctions of either protein are associated with neurological disorders. Here, we elucidate structures of the human NHE1-CHP1 complex in both inward- and inhibitor (cariporide)-bound outward-facing conformations. We find that NHE1 assembles as a symmetrical homodimer, with each subunit undergoing an elevator-like conformational change during cation exchange. The cryo-EM map reveals the binding site for the NHE1 inhibitor cariporide, illustrating how inhibitors block transport activity. The CHP1 molecule differentially associates with these two conformational states of each NHE1 monomer, and this association difference probably underlies the regulation of NHE1 pH-sensitivity by CHP1.

Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome.

Genetic screening has identified numerous variants of the endosomal solute carrier family 9 member A6 (SLC9A6)/(Na+,K+)/H+ exchanger 6 (NHE6) gene that cause Christianson syndrome, a debilitating X-linked developmental disorder associated with a range of neurological, somatic, and behavioral symptoms. Many of these variants cause complete loss of NHE6 expression, but how subtler missense substitutions or nonsense mutations that partially truncate its C-terminal cytoplasmic regulatory domain impair NHE6 activity and endosomal function are poorly understood. Here, we describe the molecular and cellular consequences of six unique mutations located in the N-terminal cytoplasmic segment (A9S), the membrane ion translocation domain (L188P and G383D), and the C-terminal regulatory domain (E547*, R568Q, and W570*) of human NHE6 that purportedly cause disease. Using a heterologous NHE6-deficient cell expression system, we show that the biochemical, catalytic, and cellular properties of the A9S and R568Q variants were largely indistinguishable from those of the WT transporter, which obscured their disease significance. By contrast, the L188P, G383D, E547*, and W570* mutants exhibited variable deficiencies in biosynthetic post-translational maturation, membrane sorting, pH homeostasis in recycling endosomes, and cargo trafficking, and they also triggered apoptosis. These findings broaden our understanding of the molecular dysfunctions of distinct NHE6 variants associated with Christianson syndrome.

A potential gain-of-function variant of SLC9A6 leads to endosomal alkalinization and neuronal atrophy associated with Christianson Syndrome.

Loss-of-function mutations in the recycling endosomal (Na+,K+)/H+ exchanger gene SLC9A6/NHE6 result in overacidification and dysfunction of endosomal-lysosomal compartments, and cause a neurodevelopmental and degenerative form of X-linked intellectual disability called Christianson Syndrome (CS). However, knowledge of the disease heterogeneity of CS is limited. Here, we describe the clinical features and underlying molecular and cellular mechanisms associated with a CS patient carrying a de novo missense variant (p.Gly218Arg; G218R) of a conserved residue in its ion translocation domain that results in a potential gain-of-function. The patient manifested several core symptoms typical of CS, including pronounced cognitive impairment, mutism, epilepsy, ataxia and microcephaly; however, deterioration of motor function often observed after the first decade of life in CS children with total loss of SLC9A6/NHE6 function was not evident. In transfected non-neuronal cells, complex glycosylation and half-life of the G218R were significantly decreased compared to the wild-type transporter. This correlated with elevated ubiquitination and partial proteasomal-mediated proteolysis of G218R. However, a major fraction was delivered to the plasma membrane and endocytic pathways. Compared to wild-type, G218R-containing endosomes were atypically alkaline and showed impaired uptake of recycling endosomal cargo. Moreover, instead of accumulating in recycling endosomes, G218R was redirected to multivesicular bodies/late endosomes and ejected extracellularly in exosomes rather than progressing to lysosomes for degradation. Attenuated acidification and trafficking of G218R-containing endosomes were also observed in transfected hippocampal neurons, and correlated with diminished dendritic branching and density of mature mushroom-shaped spines and increased appearance of filopodia-like protrusions. Collectively, these findings expand our understanding of the genetic diversity of CS and further elucidate a critical role for SLC9A6/NHE6 in fine-tuning recycling endosomal pH and cargo trafficking, processes crucial for the maintenance of neuronal polarity and mature synaptic structures.

Efficacy of hybrid vitamin D receptor agonist/histone deacetylase inhibitors in vitamin D-resistant triple-negative 4T1 breast cancer.

Hormonal 1,25-dihydroxyvitamin D (1,25D) and its analogues have shown efficacy in some preclinical models of cancer. However, many models are resistant to the antiproliferative effects of 1,25D or its analogues in vitro or in vivo, and such compounds have failed in the clinic as monotherapies because of tumor resistance. Given the observed synergism between 1,25D analogues and histone deacetylase inhibitors (HDACi) in 1,25D-resistant cells, we previously developed a series of hybrid secosteroidal and easily assembled non-secosteroidal analogues that combined agonism for the vitamin D receptor and HDACi in a single backbone. These compounds displayed enhanced efficacy against 1,25D-resistant malignant cells in vitro. Structure/function studies led to synthesis of several non-secosteroidal variants in which HDACi potency was optimized without substantially sacrificing VDR agonism. Here, we present the first studies of efficacy in vivo of two of these compounds, DK-366 and DK-406, in the aggressive mouse 4T1 model of triple-negative breast cancer, a form of the disease for which treatment options are limited. 4T1 cells are resistant in vitro to the cytostatic and cytotoxic effects of 1,25D and the potent HDACi SAHA individually up to concentrations of 1µM and 50µM, respectively, whereas combinations of the two are efficacious. In vitro, DK-366 or -406 induced dose-dependent arrest of cell proliferation and cytotoxicity at 10-20µM. In vivo, the maximum tolerated dose (MTD) of DK-366 and DK-406 were 2.5 and 5.0mg/kg, respectively. Although the compounds induced hypercalcemia at elevated doses, consistent with VDR agonism in vivo, they both reduced tumor burden at doses below their MTD's. Moreover, in a separate experiment, DK-406 at 5mg/kg reduced 4T1 lung metastases by at least 50%. Under the same conditions, 1,25D (0.25µg/kg) and SAHA (25mg/kg) combined had no effect on tumor burden or on lung metastases. These experiments show that hybrid compounds are bioavailable and efficacious against a particularly aggressive model of metastatic breast cancer, providing strong support for the therapeutic potential of the hybrid concept.

A Christianson syndrome-linked deletion mutation (∆(287)ES(288)) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death.

BACKGROUND: Christianson Syndrome, a recently identified X-linked neurodevelopmental disorder, is caused by mutations in the human gene SLC9A6 encoding the recycling endosomal alkali cation/proton exchanger NHE6. The patients have pronounced limitations in cognitive ability, motor skills and adaptive behaviour. However, the mechanistic basis for this disorder is poorly understood as few of the more than 20 mutations identified thus far have been studied in detail. METHODS: Here, we examined the molecular and cellular consequences of a 6 base-pair deletion of amino acids Glu(287) and Ser(288) (∆ES) in the predicted seventh transmembrane helix of human NHE6 expressed in established cell lines (CHO/AP-1, HeLa and neuroblastoma SH-SY5Y) and primary cultures of mouse hippocampal neurons by measuring levels of protein expression, stability, membrane trafficking, endosomal function and cell viability. RESULTS: In the cell lines, immunoblot analyses showed that the nascent mutant protein was properly synthesized and assembled as a homodimer, but its oligosaccharide maturation and half-life were markedly reduced compared to wild-type (WT) and correlated with enhanced ubiquitination leading to both proteasomal and lysosomal degradation. Despite this instability, a measurable fraction of the transporter was correctly sorted to the plasma membrane. However, the rates of clathrin-mediated endocytosis of the ∆ES mutant as well as uptake of companion vesicular cargo, such as the ligand-bound transferrin receptor, were significantly reduced and correlated with excessive endosomal acidification. Notably, ectopic expression of ∆ES but not WT induced apoptosis when examined in AP-1 cells. Similarly, in transfected primary cultures of mouse hippocampal neurons, membrane trafficking of the ∆ES mutant was impaired and elicited marked reductions in total dendritic length, area and arborization, and triggered apoptotic cell death. CONCLUSIONS: These results suggest that loss-of-function mutations in NHE6 disrupt recycling endosomal function and trafficking of cargo which ultimately leads to neuronal degeneration and cell death in Christianson Syndrome.

Determinants of Cation Permeation and Drug Sensitivity in Predicted Transmembrane Helix 9 and Adjoining Exofacial Re-entrant Loop 5 of Na+/H+ Exchanger NHE1.

Mammalian Na(+)/H(+) exchangers (NHEs) regulate numerous physiological processes and are involved in the pathogenesis of several diseases, including tissue ischemia and reperfusion injuries, cardiac hypertrophy and failure, and cancer progression. Hence, NHEs are being targeted for pharmaceutical-based clinical therapies, but pertinent information regarding the structural elements involved in cation translocation and drug binding remains incomplete. Molecular manipulations of the prototypical NHE1 isoform have implicated several predicted membrane-spanning (M) helices, most notably M4, M9, and M11, as important determinants of cation permeation and drug sensitivity. Here, we have used substituted-cysteine accessibility mutagenesis and thiol-modifying methanethiosulfonate (MTS) reagents to further probe the involvement of evolutionarily conserved sites within M9 (residues 342-363) and the adjacent exofacial re-entrant loop 5 between M9 and M10 (EL5; residues 364-415) of a cysteine-less variant of rat NHE1 on its kinetic and pharmacological properties. MTS treatment significantly reduced the activity of mutants containing substitutions within M9 (H353C, S355C, and G356C) and EL5 (G403C and S405C). In the absence of MTS, mutants S355C, G403C, and S405C showed modest to significant decreases in their apparent affinities for Na(+) o and/or H(+) i. In addition, mutations Y370C and E395C within EL5, whereas failing to confer sensitivity to MTS, nevertheless, reduced the affinity for Na(+) o, but not for H(+) i. The Y370C mutant also exhibited higher affinity for ethylisopropylamiloride, a competitive antagonist of Na(+) o transport. Collectively, these results further implicate helix M9 and EL5 of NHE1 as important elements involved in cation transport and inhibitor sensitivity, which may inform rational drug design.

Impaired posttranslational processing and trafficking of an endosomal Na+/H+ exchanger NHE6 mutant (Δ(370)WST(372)) associated with X-linked intellectual disability and autism.

Na(+)/H(+) exchanger NHE6/SLC9A6 is an X-linked gene that is widely expressed and especially abundant in brain, heart and skeletal muscle where it is implicated in endosomal pH homeostasis and trafficking as well as maintenance of cell polarity. Recent genetic studies have identified several mutations in the coding region of NHE6 that are linked with severe intellectual disability, autistic behavior, ataxia and other abnormalities. One such defect consists of an in-frame deletion of three amino acids ((370)Trp-Ser-Thr(372), ΔWST) that adjoin the predicted ninth transmembrane helix of the exchanger. To better understand the nature of this mutation, a NHE6ΔWST construct was generated and assessed for its effects on the biochemical and cellular properties of the transporter. In transfected fibroblastic CHO and neuroblastoma SH-SY5Y cells, immunoblot analyses showed that the mutant protein was effectively synthesized, but its subsequent oligosaccharide maturation and overall half-life were dramatically reduced compared to wild-type. These changes correlated with significant accumulation of ΔWST in the endoplasmic reticulum, with only minor sorting to the plasma membrane and negligible trafficking to recycling endosomes. The diminished accumulation in recycling endosomes was associated with a significant decrease in the rate of endocytosis of cell surface ΔWST compared to wild-type. Furthermore, while ectopic expression of wild-type NHE6 enhanced the uptake of other vesicular cargo such as transferrin along the clathrin-mediated recycling endosomal pathway, this ability was lost in the ΔWST mutant. Similarly, in transfected primary mouse hippocampal neurons, wild-type NHE6 was localized in discrete puncta throughout the soma and neurites, whereas the ΔWST mutant displayed a diffuse reticular pattern. Remarkably, the extensive dendritic arborization observed in neurons expressing wild-type NHE6 was noticeably diminished in ΔWST-transfectants. These results suggest that deletion of (370)Trp-Ser-Thr(372) leads to endoplasmic reticulum retention and loss of NHE6 function which potentially impacts the trafficking of other membrane-bound cargo and cell polarity.

Genetic diversity of the golden potato cyst nematode Globodera rostochiensis and determination of the origin of populations in Quebec, Canada.

The golden cyst nematode (Globodera rostochiensis), native to South America, has been introduced in many parts of the world, including Europe and North America. Recently, it was found for the first time in the province of Quebec, Canada in the locality of St. Amable near Montreal. To date, very few studies have examined the population genetics of this pest. Consequently, there is a lack of knowledge about the genetic structure and evolution of this nematode. In this study, twelve new microsatellite markers were developed in order to explore these questions. These markers were used to genotype fifteen populations originating from different regions of the world, including five from Canada. Within populations, the highest genetic diversity was consistently observed in the populations from Bolivia, the postulated region of origin of the golden nematode, and the lowest in populations from British Columbia (Canada) and New York (USA). The two Quebec populations were very similar to each other and to the population found in Newfoundland, but surprisingly, they were significantly different from three other North American populations including those from New York and British Columbia. Based on our results, we conclude that the golden cyst nematode has been introduced in North America at least twice from distinct regions of the world.

Membrane surface charge dictates the structure and function of the epithelial Na+/H+ exchanger.

The Na(+)/H(+) exchanger NHE3 plays a central role in intravascular volume and acid-base homeostasis. Ion exchange activity is conferred by its transmembrane domain, while regulation of the rate of transport by a variety of stimuli is dependent on its cytosolic C-terminal region. Liposome- and cell-based assays employing synthetic or recombinant segments of the cytosolic tail demonstrated preferential association with anionic membranes, which was abrogated by perturbations that interfere with electrostatic interactions. Resonance energy transfer measurements indicated that segments of the C-terminal domain approach the bilayer. In intact cells, neutralization of basic residues in the cytosolic tail by mutagenesis or disruption of electrostatic interactions inhibited Na(+)/H(+) exchange activity. An electrostatic switch model is proposed to account for multiple aspects of the regulation of NHE3 activity.

No-needle jet intradermal aminolevulinic Acid photodynamic therapy for recurrent nodular Basal cell carcinoma of the nose: a case report.

Photodynamic therapy (PDT) with aminolevulinic acid (ALA) to treat nodular basal cell carcinoma (BCC) has been shown to be beneficial. The success rate of ALA-PDT in the treatment of nodular BCC is dependent on optimal penetration of the photosensitizing agent and subsequent PpIX production. To enhance topical delivery of drugs intradermally, a needleless jet injection (NLJI), which employs a high-speed jet to puncture the skin without the side effects of needles, was used in one patient with recurrent BCC of the nose. Photoactivation was then performed using red light emitting diode [CW @ λ 630 nm, irradiance 50 mW/cm(2), total fluence 51 J/cm(2)] for 17 minutes. Excellent cosmesis was obtained. Aside from mild crusting present for six days, no other adverse signs were noted. Clinically, there was no recurrent lesion up two years postintervention. Additional studies in larger samples of subjects are needed to further evaluate this promising technique.

Prophylactic low-level light therapy for the treatment of hypertrophic scars and keloids: a case series.

BACKGROUND AND OBJECTIVES: Hypertrophic and keloid scars result from alterations in the wound healing process. Treating abnormal scars remains an important challenge. The aim of this case series was to investigate the effectiveness of near infrared (NIR) light emitting diode (LED) treatment as a prophylactic method to alter the wound healing process in order to avoid or attenuate the formation of hypertrophic scars or keloids. STUDY DESIGN/PATIENTS AND METHODS: Three patients (age 27-57) of phototypes I-III with hypertrophic scars or keloids due to acne or surgery participated in this case series. Following scar revision by surgery or CO(2) laser ablation on bilateral areas, one scar was treated daily by the patient at home with non-thermal, non-ablative NIR LED (805 nm at 30 mW/cm(2)) for 30 days. Efficacy assessments, conducted up to a year post-treatment, included the Vancouver Scar scale (VSS), clinical global assessment of digital photographs, and quantitative profilometry analysis using PRIMOS. Safety was documented by adverse effects monitoring. RESULTS: Significant improvements on the NIR-treated versus the control scar were seen in all efficacy measures. No significant treatment-related adverse effects were reported. CONCLUSION: Possible mechanisms involved are inhibition of TGF-beta I expression. Further studies in larger group of patients are needed to evaluate this promising technique.

Radiant near infrared light emitting Diode exposure as skin preparation to enhance photodynamic therapy inflammatory type acne treatment outcome.

BACKGROUND AND OBJECTIVE: An alternative approach in the treatment of acne vulgaris is photodynamic therapy (PDT) that uses light and aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) production to eradicate Propionibacterium acnes found in acne lesions. PpIX formation is dependent on ALA percutaneaous penetration. In this study, to enhance ALA penetration and subsequent accumulation of PpIX, skin temperature was increased with radiant infrared (IR) prior to ALA-PDT application and compared to ALA-PDT alone in the treatment of inflammatory acne. STUDY DESIGN/MATERIALS AND METHODS: Ten patients exhibiting inflammatory acne with a lesion count of > or =10 were assigned to a split face or split back group. One side was pre-treated for 15 minutes with radiant IR light emitting diode (LED) (970 nm), while the other side was used as control. ALA was then applied after which PDT LED (630 nm) was performed on the entire face or back surface. Blinded lesion counts and clinical global assessment of severity were performed based on digital photographs before and 4 weeks after the PDT procedure. RESULTS: This randomized, controlled, and rater-blinded trial revealed a significant difference in median reduction of inflammatory lesions on the IR pre-treated (73%, 95% confidence interval (CI) 51-81%) versus the control side (38%, 95% CI 8-55%) 1 month after PDT (P<0.0001). Clinical assessment of severity was also significantly lower on the IR-treated side than on the control side (median 1, 95% CI 0.74-1.34 vs. 2, 95% CI 1.17-1.72). No unusual treatment-related adverse effects were observed. CONCLUSION: The reported therapeutic effects may be due to enhanced induction of alterations in transcutaneous diffusion kinetics of the photosensitizer at higher skin temperature and/or conversion of ALA to PpIX. Pre-PDT radiant IR LED exposure appears to be a promising method to enhance PDT efficacy for the treatment of acne lesions.

Regulation of skin collagen metabolism in vitro using a pulsed 660 nm LED light source: clinical correlation with a single-blinded study.

It has been reported that skin aging is associated with a downregulation in collagen synthesis and an elevation in matrix metalloproteinase (MMP) expression. This study investigated the potential of light-emitting diode (LED) treatments with a 660 nm sequentially pulsed illumination formula in the photobiomodulation of these molecules. Histological and biochemical changes were first evaluated in a tissue-engineered Human Reconstructed Skin (HRS) model after 11 sham or LED light treatments. LED effects were then assessed in aged/photoaged individuals in a split-face single-blinded study. Results yielded a mean percent difference between LED-treated and non-LED-treated HRS of 31% in levels of type-1 procollagen and of -18% in MMP-1. No histological changes were observed. Furthermore, profilometry quantification revealed that more than 90% of individuals showed a reduction in rhytid depth and surface roughness, and, via a blinded clinical assessment, that 87% experienced a reduction in the Fitzpatrick wrinkling severity score after 12 LED treatments. No adverse events or downtime were reported. Our study showed that LED therapy reversed collagen downregulation and MMP-1 upregulation. This could explain the improvements in skin appearance observed in LED-treated individuals. These findings suggest that LED at 660 nm is a safe and effective collagen-enhancement strategy.

LED photoprevention: reduced MED response following multiple LED exposures.

BACKGROUND AND OBJECTIVES: As photoprotection with traditional sunscreen presents some limitations, the use of non-traditional treatments to increase skin resistance to ultraviolet (UV) induced damage would prove particularly appealing. The purpose of this pilot study was to test the potential of non-thermal pulsed light-emitting diode (LED) treatments (660 nm) prior to UV exposure in the induction of a state of cellular resistance against UV-induced erythema. STUDY DESIGN/MATERIALS AND METHODS: Thirteen healthy subjects and two patients with polymorphous light eruption (PLE) were exposed to 5, 6, or 10 LED treatments (660 nm) on an EXPERIMENTAL anterior thigh region. Individual baseline minimal erythema doses (MED) were then determined. UV radiation was thereafter performed on the LED EXPERIMENTAL and CONTROL anterior thigh areas. Finally, 24 hours post-UV irradiation, LED pre-treated MED responses were compared to the non-treated sites. RESULTS: Reduction of erythema was considered significant when erythema was reduced by >50% on the LED-treated side as opposed to CONTROL side. A significant LED treatment reduction in UV-B induced erythema reaction was observed in at least one occasion in 85% of subjects, including patients suffering from PLE. Moreover, there was evidence of a dose-related pattern in results. Finally, a sun protection factor SPF-15-like effect and a reduction in post-inflammatory hyperpigmentation were observed on the LED pre-treated side. CONCLUSIONS: Results suggest that LED based therapy prior to UV exposure provided significant protection against UV-B induced erythema. The induction of cellular resistance to UV insults may possibly be explained by the induction of a state a natural resistance to the skin via specific cell signaling pathways and without the drawbacks and limitations of traditional sunscreens. These results represent an encouraging step towards expanding the potential applications of LED therapy and could be useful in the treatment of patients with anomalous reactions to sunlight.

Long-term human coronavirus-myelin cross-reactive T-cell clones derived from multiple sclerosis patients.

Autoimmune reactions associated with MS involve genetic and environmental factors. Because murine coronaviruses induce an MS-like disease, the human coronaviruses (HCoV) are attractive candidates as environmental factors involved in a demyelinating pathology. We previously reported the isolation of HCoV-229E/myelin basic protein (MBP) cross-reactive T-cell lines (TCL) in MS patients. To investigate antigenic cross-reactivity at the molecular level, 155 long-term T-cell clones (TCC) were derived from 32 MS patients by in vitro selection with MBP, proteolipid protein (PLP) or HCoV (strains 229E and OC43). Overall, 114 TCC were virus-specific, 31 were specific for myelin Ag and 10 other were HCoV/myelin cross-reactive. Twenty-eight virus-specific TCC and 7 myelin-specific TCC were obtained from six healthy donors. RACE RT-PCR amplification of the Vbeta chains of five of ten the cross-reactive TCC confirmed clonality and sequencing identified the CDR3 region associated with cross-reactivity. Our findings have promising implications in the investigation of the role of molecular mimicry between coronaviruses and myelin in MS as a mechanism related to disease initiation or relapses.