Photoaffinity Labeling of Nicotinic Receptors: Diversity of Drug Binding Sites!

For almost 30 years, photoaffinity labeling and protein microsequencing techniques have been providing novel insights about the structure of nicotinic acetylcholine receptors (nAChR) and the diversity of nAChR drug binding sites. Photoaffinity labeling allows direct identification of amino acid residues contributing to a drug binding site without prior knowledge of the location of the binding site within the nAChR or the orientation of the ligand within the binding site. It also distinguishes amino acids that contribute to allosteric binding sites from those involved in allosteric modulation of gating. While photoaffinity labeling was used initially to identify amino acids contributing to the agonist binding sites and the ion channel, it has been used recently to identify binding sites for allosteric modulators at subunit interfaces in the extracellular and the transmembrane domains, and within a subunit's transmembrane helix bundle. In this article, we review the different types of photoaffinity probes that have been used and the various binding sites that have been identified within the structure of nAChR, with emphasis on our recent studies of allosteric modulator binding sites.     

ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress

Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson’s disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6. These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

Loss-of-function mutations in ATP13A2 (PARK9) are causative for a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome (KRS, a juvenile onset parkinsonism with dementia) (1), early-onset Parkinson’s disease (PD) (2, 3), hereditary spastic paraplegia (HSP) (4), neuronal ceroid lipofuscinosis (5), and amyotrophic lateral sclerosis (6), which are commonly hallmarked by lysosomal and mitochondrial dysfunction (4, 6, 7). Also, ATP13A2 deficiency causes lysosomal and mitochondrial impairment in various models, as evidenced by decreased lysosomal functionality (8, 9), reduced mitochondrial clearance capacity (8–10), mitochondrial fragmentation, mitochondrial DNA damage, and increased oxygen consumption (11, 12).We recently discovered that ATP13A2 transports the polyamines spermidine and spermine from the late endo/lysosome to the cytosol (9). Polyamines are ubiquitous polycationic aliphatic amines that stabilize nucleic acids, influence protein folding, regulate ion channels, and modulate cell proliferation and differentiation (13–15). We found that the late endo-lysosomal transporter ATP13A2 strongly contributes to the total cellular polyamine content via a two-step process: Firstly, polyamines enter the cell via endocytosis and subsequently, polyamines are transported by ATP13A2 into the cytosol (9). This process complements polyamine biosynthesis via the ornithine decarboxylase (ODC) pathway (9). Importantly, ATP13A2’s polyamine transport function is crucial for its neuroprotective effect, since it prevents lysosomal polyamine accumulation and subsequent lysosomal rupture, while improving lysosomal health and functionality (9). Moreover, when activated by its two regulatory lipids—phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] and phosphatidic acid (PA)—ATP13A2 exerts a cell protective effect against the mitochondrial neurotoxin rotenone (16), an environmental risk factor for PD (17). Rotenone is a mitochondrial complex I inhibitor, which leads to high levels of reactive oxygen species (ROS), promoting protein aggregation and damaging organelles. However, how ATP13A2’s polyamine transport function exerts a cell protective effect against rotenone, or other mitochondrial neurotoxins, is not yet clear.Interestingly, the transported substrates spermine and spermidine reduce oxidative stress (14, 15). Spermine is a potent free radical scavenger (18) and a biologically important antioxidant (19–23). We therefore hypothesize that ATP13A2-mediated polyamine transport may counteract oxidative stress (16, 24) and preserve mitochondrial health (11, 12). Here, we demonstrate in complementary human cell models and Caenorhabditis elegans that lysosomal polyamine export by ATP13A2 effectively lowers ROS levels and promotes mitochondrial health and functionality, pointing to a lysosomal-dependent cell protective pathway that may be implicated in ATP13A2-related neurodegenerative disorders.     

Statistical optimization of photo-induced biofabrication of silver nanoparticles using the cell extract of Oscillatoria limnetica: insight on characterization and antioxidant potentiality

Silver nanoparticles were successfully fabricated through a very simple, rapid, one-step photo-induced green approach. The formation of silver nanoparticles was accomplished using the bioactive compounds in the aqueous extract of fresh Oscillatoria limnetica biomass, which acted as a reducing and capping agent at the same time. The biosynthesis of Oscillatoria-silver nanoparticles (O-AgNPs) was investigated under the influence of different light intensities 57.75, 75.90 and 1276.51 μmol m⁻² s⁻¹ (bright sunlight). UV-Vis (UV) and Fourier transform infrared (FT-IR) spectroscopy were applied to approve the synthesis of AgNPs. Further, the synthesis process under the exposure to sunlight was adjusted via utilizing one factor at a time, and 0.5 mM AgNO₃ concentration, 5 mL O. limnetica solution, pH 6.7 and 30 min sunlight (1276.51 μmol m⁻² s⁻¹) were applied. Furthermore, the central composite design (CCD) was applied to boost the biosynthesis process of O-AgNPs (manufactured at light intensity 75.90 μmol m⁻² s⁻¹). The maximum production of O-AgNPs was attained with 4 detected variables: initial pH level (6.7), AgNO₃ concentration (0.3 mM), O. limnetica extract concentration (3.50 mL) and incubation time (48 h). Moreover, TEM, in addition to SEM, images exposed that the biosynthesized AgNPs were quasi-spherical in shape with a small monodisperse nature, and the size range was between 6.98–23.48 nm in the case of light-induced synthesis (75.90 μmol m⁻² s⁻¹) and 11.58–22.31 nm with sunlight (1276.51 μmol m⁻² s⁻¹).

Silver nanoparticles were successfully fabricated through a very simple, rapid, one-step photo-induced green approach.     

Cystic echinococcosis in children in Tunisia: fertility and case distribution of hydatid cysts

Cystic echinococcosis, which commonly starts during childhood or adolescence, is a serious problem of public health in Tunisia. For 121 children (161 cysts), the localization and fertility of cysts as well as viability of their protoscoleces were determined. Results indicated that the lung was the primary localization of cyst (59%) followed by the liver (35%). Children's infection is more frequent in male than in female (sex ratio 1.96) and the greatest number of cases is observed in the 4-9 year age groups (94 cases). The fertility of the cyst was independent of its site or its size and no incidence of age of children was detected. Nevertheless, the fertility rate is higher in females than in males for the liver localization.     

West syndrome associated with Down syndrome: Case report and literature review

West syndrome is the most frequent cause of epilepsy in Down syndrome. West syndrome is often associated with poor long-term prognosis in most of children. We report a girl with West syndrome associated with Down syndrome which occurred at 8 months of age for repetitive flexor spasms and electroencephalography (EEG) showed hypsarrhythmia. She had Down syndrome facies, microcephaly, psychomotor development delay and axial hypotonia. Computed tomography of the brain was normal. Her karyotype was 47, XX, +21. Phenobarbital therapy was immediately effective with good clinical control of seizures, while the EEG monitored after one month was unchanged. At 2 years of age, the patient had hypertonic status epilepticus following a lung infection. The EEG showed a persistence of hypsarrhythmia. Sodium valproate and hydrocortisone therapy was effective with good seizure control but her psychomotor development was severely impaired. After a follow-up of 7 years, the patient presents growth retardation, microcephaly, severe psychomotor development delay, generalized hypotonia and tetraparesis. Knowledge of West syndrome in Down syndrome allows the early detection and prompt management of this neurological complication in order to optimize psychomotor development and improve the quality of life of these children.     

Genetic polymorphism of glutathione S-transferases M1 and T1 in Egyptian patients with bilharzial bladder cancer

ObjectiveTo assess the influence of glutathione S-transferases M1 and T1 (GSTM1 and T1) genotype on the risk of bladder cancer in patients with urinary bilharziasis.Materials and methodsThis study was designed as a case-control study that involved 60 individuals who were enrolled into 3 equal groups. The first one included patients with bilharzial bladder cancer, the second one had those with nonmalignant urinary bilharziasis, and the last one was the control group. All of the participants were adult males, nonsmokers, and with matched ages. All of them underwent an assessment of the serum level of the total GST concentration and the polymerase chain reaction (PCR) was used for determination of the GSTM1 and T1 genotypes.ResultsThe lower most GST enzyme concentration was reported in patients with bilharzial bladder cancer (26 ± 4.4 ng/ml) with significant difference between it and that of the second group (36.8 ± 4.1 ng/ml, P < 0.05) and that of the controls (40.4 ± 4 ng/ml, P < 0.005). The PCR results have demonstrated that the frequency of combined GSTM1 and T1 genes deletion (M1–ve T1–ve) was significantly higher in cases of bladder cancer (40%) than those of the controls (5%, P < 0.005) and those of the second group (10%, P < 0.05). The unconditional logistic regression test revealed that patients with urinary bilharziasis and combined GSTM1 and T1 genes deletion are at a significant risk for malignant transformation (OR = 6.3, P < 0.05).ConclusionsPatients with urinary bilharziasis and GSTM1–ve and T1–ve genes might be at increased risk of bladder cancer. However, larger studies are needed for confirmation of these results.