Co– and Post–Translational N–Linked Glycosylation of Cardiac Potassium Channel Subunits: A Dissertation

KCNE1 (E1) peptide is the founding member of the KCNE family (1-5), which is a class of type I transmembrane ß-subunits. KCNE1 peptides assemble with and modulate the gating, ion conducting properties and pharmacology of a variety of voltage-gated K+ channel a-subunits, including KCNQ1 (Q1). Mutations that interfere with the function of either E1 and/or Q1 and disrupt the assembly and trafficking of KCNE1- KCNQ1 channel complexes give rise to diseases such as Romano-Ward (RW) and Jervell Lange Nielsen Syndrome (JLNS), two different forms of Long QT Syndrome (LQTS). Using enzymatic deglycosylation assays, immunofluorescence techniques and quantitative cell surface labeling, we showed that KCNE1 peptides are retained in the early stages of the secretory pathway as immaturely N-linked glycosylated proteins. KCNE1 co-assembly with KCNQ1 leads to E1 progression through the secretory pathway and glycan maturation, resulting in cell surface expression. N-linked glycosylation of some membrane proteins is critical for proper folding, co-assembly and subsequent trafficking through the biosynthetic pathway. Previous studies have shown that genetic mutations that disrupt one of the two N-linked glycosylation sites on KCNE family members lead to LQTS (T7I, KCNE1 and T8A, KCNE2) (Schulze-Bahr et al., 1997; Sesti et al., 2000a; Park et al., 2003). Having confirmed that KCNE1 proteins acquire N-linked glycans, we examined the kinetics and efficiency of N-linked glycan addition to KCNE1. We showed that KCNE1 has two distinct N-linked glycosylation sites. The N-terminal sequon is a traditional co-translational site. The internal sequon (which is only ~ 20 residues away from the N-terminal sequon) acquires N-linked glycans primarily after protein synthesis (post-translationally). Surprisingly, mutations that prevent N-glycosylation at the cotranslational site also reduce the glycosylation efficiency of post-translational glycosylation at the internal sequon, resulting in a large population of unglycosylated KCNE1 peptides that are retained in the early stages of the secretory pathway and do not reach the cell surface with their cognate K+ channel. We showed that KCNE1 post-translational N-glycosylation in the endoplasmic reticulum is a cellular mechanism that ensures E1 proteins acquire the maximal number of glycans needed for proper channel assembly and trafficking. Our findings provide a new biogenic mechanism for human disease by showing that the JLNS mutation, T7I, not only inhibits glycosylation of the N-terminal sequon, but also indirectly prevents the glycosylation of the internal sequon, giving rise to a large population of assembly incompetent hypoglycosylated KCNE1 peptides. To further investigate the two N-linked glycosylation sites on KCNE1, we generated structure-function deletion scans of KCNE1 and performed positional glycosylation scanning mutagenesis. We examined the glycosylation pattern of glycosylation mutants in an effort to define the glycosylation window important for proper KCNE1 assembly and trafficking. Our findings suggested a nine amino acid periodicity to serve as a desirable glycosylation site and a better substrate for N-glycosylation. Appendix II shows work on the characterization of the C-terminally HA-tagged KCNE1 protein, which was used throughout the experiments presented in Chapter II, Chapter III and Chapter IV. Analysis of the C-terminally HA-tagged KCNE1 protein revealed that in heterologous expression systems KCNE1 had an internal translational start site, a methionine at position 27. A proteolytic cleavage site was also identified at the arginine cluster spanning residues 32 through 38 bearing the two known Long QT mutations (R32H and R36H) (Splawski et al., 2000; Napolitano et al., 2005). My work in Professor Craig C. Mello’s lab during the first four years of my graduate study is presented in Appendix I. The highly conserved Wnt/Wingless glycoproteins regulate many aspects of animal development. Wnt signaling specifies endoderm fate by controlling the fate of EMS blastomere daughters in 4-cell stage Caenorhabditis elegans embryos. A suppressor genetic screen was performed using two temperature sensitive alleles of mom-2/Wnt to identify additional regulators of the Wnt/Wingless signaling pathway during C. elegans endoderm specification. Five intragenic suppressors and three extragenic suppressors of mom-2/Wnt embryonic lethality were identified. We cloned ifg-1, eIF4G homologue, as one of the extragenic suppressors suggesting an intriguing connection between the Wnt signaling pathway and the translational machinery

Long-Term Outcomes With Nivolumab Plus Ipilimumab or Nivolumab Alone Versus Ipilimumab in Patients With Advanced Melanoma

PURPOSE In the phase III CheckMate 067 trial, durable clinical benefit was demonstrated previously with nivolumab plus ipilimumab and nivolumab alone versus ipilimumab. Here, we report 6.5-year efficacy and safety outcomes. PATIENTS AND METHODS Patients with previously untreated unresectable stage III or stage IV melanoma were randomly assigned 1:1:1 to receive nivolumab 1 mg/kg plus ipilimumab 3 mg/kg once every 3 weeks (four doses) followed by nivolumab 3 mg/kg once every 2 weeks (n = 314), nivolumab 3 mg/kg once every 2 weeks (n = 316), or ipilimumab 3 mg/kg once every 3 weeks (four doses; n = 315). Coprimary end points were progression-free survival and overall survival (OS) with nivolumab plus ipilimumab or nivolumab versus ipilimumab. Secondary end points included objective response rate, descriptive efficacy assessments of nivolumab plus ipilimumab versus nivolumab alone, and safety. Melanoma-specific survival (MSS; descriptive analysis), which excludes deaths unrelated to melanoma, was also evaluated. RESULTS Median OS (minimum follow-up, 6.5 years) was 72.1, 36.9, and 19.9 months in the combination, nivolumab, and ipilimumab groups, respectively. Median MSS was not reached, 58.7, and 21.9 months, respectively; 6.5-year OS rates were 57%, 43%, and 25% in patients with BRAF-mutant tumors and 46%, 42%, and 22% in those with BRAF–wild-type tumors, respectively. In patients who discontinued treatment, the median treatment-free interval was 27.6, 2.3, and 1.9 months, respectively. Since the 5-year analysis, no new safety signals were observed. CONCLUSION These 6.5-year CheckMate 067 results, which include the longest median OS in a phase III melanoma trial reported to date and the first report of MSS, showed durable, improved clinical outcomes with nivolumab plus ipilimumab or nivolumab versus ipilimumab in patients with advanced melanoma and, in descriptive analyses, with the combination over nivolumab monotherapy

Adjuvant Therapy of Nivolumab Combined With Ipilimumab Versus Nivolumab Alone in Patients With Resected Stage IIIB-D or Stage IV Melanoma (CheckMate 915)

PURPOSE Ipilimumab and nivolumab have each shown treatment benefit for high-risk resected melanoma. The phase III CheckMate 915 trial evaluated adjuvant nivolumab plus ipilimumab versus nivolumab alone in patients with resected stage IIIB-D or IV melanoma. PATIENTS AND METHODS In this randomized, double-blind, phase III trial, 1,833 patients received nivolumab 240 mg once every 2 weeks plus ipilimumab 1 mg/kg once every 6 weeks (916 patients) or nivolumab 480 mg once every 4 weeks (917 patients) for <= 1 year. After random assignment, patients were stratified by tumor programmed death ligand 1 (PD-L1) expression and stage. Dual primary end points were recurrence-free survival (RFS) in randomly assigned patients and in the tumor PD-L1 expression-level < 1% subgroup. RESULTS At a minimum follow-up of approximately 23.7 months, there was no significant difference between treatment groups for RFS in the all-randomly assigned patient population (hazard ratio, 0.92; 95% CI, 0.77 to 1.09; P = .269) or in patients with PD-L1 expression < 1% (hazard ratio, 0.91; 95% CI, 0.73 to 1.14). In all patients, 24-month RFS rates were 64.6% (combination) and 63.2% (nivolumab). Treatment-related grade 3 or 4 adverse events were reported in 32.6% of patients in the combination group and 12.8% in the nivolumab group. Treatment-related deaths were reported in 0.4% of patients in the combination group and in no nivolumab-treated patients. CONCLUSION Nivolumab 240 mg once every 2 weeks plus ipilimumab 1 mg/kg once every 6 weeks did not improve RFS versus nivolumab 480 mg once every 4 weeks in patients with stage IIIB-D or stage IV melanoma. Nivolumab showed efficacy consistent with previous adjuvant studies in a population resembling current practice using American Joint Committee on Cancer eighth edition, reaffirming nivolumab as a standard of care for melanoma adjuvant treatment

KCNE1 subunits require co-assembly with K+ channels for efficient trafficking and cell surface expression

KCNE peptides are a class of type I transmembrane beta subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K(+) channels. Accordingly, mutations that disrupt the assembly and trafficking of KCNE-K(+) channel complexes give rise to disease. The cellular mechanisms responsible for ensuring that KCNE peptides assemble with voltage-gated K(+) channels have yet to be elucidated. Using enzymatic deglycosylation, immunofluorescence, and quantitative cell surface labeling experiments, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with specific K(+) channel subunits; co-assembly mediates KCNE1 progression through the secretory pathway and results in cell surface expression. We also address an apparent discrepancy between our results and a previous study in human embryonic kidney cells, which showed wild type KCNE1 peptides can reach the plasma membrane without exogenously expressed K(+) channel subunits. By comparing KCNE1 trafficking in three cell lines, our data suggest that the errant KCNE1 trafficking observed in human embryonic kidney cells may be due, in part, to the presence of endogenous voltage-gated K(+) channels in these cells

Differential effects of clozapine and risperidone on facial emotion recognition ability in patients with treatment-resistant schizophrenia

Objective: Clozapine and risperidone are used for treatment-resistant schizophrenia and known to improve the positive and negative symptoms. However, there are some conflicts about effects on social cognition, which is measured with facial emotion recognition ability. The impairments in facial emotion recognition ability have frequently been in different stages of the illness and might have negative influences on psychosocial functioning. In the present study, we aimed to examine clozapine and risperidone effects recognizing facial emotions in patient with treatment-resistant schizophrenia. Methods: Thirty-four patients were screened for the study, and 19 patients were included. All patients were evaluated with Positive and Negative Syndrome Scale (PANSS), Calgary Depression Scale for Schizophrenia, and Functional Remission of General Schizophrenia Scale at baseline and after 16–20 weeks of clozapine (n = 12) or risperidone (n = 7) treatment. Furthermore, the Facial Emotion Recognition Test was performed before and after treatment. The test included the photos of four male and four female models (totally 56 mixed photos) with happy, surprised, fearful, sad, angry, disgusted, and neutral facial expressions from Ekman and Friesen’s catalog. Results: The mean dose of the index drug in clozapine group was 295.83 ± 103.26 mg/day. The mean positive (p = .002), negative (p = .050) general psychopathology (p = .002), and total score (p = .002) according to the PANSS were significantly improved after treatment. The mean dose of the index drug in risperidone group was 6.86 ± 1.57 mg/day. The mean positive symptom (p = .018) and total score (p = .041) were significantly improved after treatment but negative symptom scale (p = .396) and general psychopathology (p = .149) scores did not change. There were no significant differences between baseline and after treatment in clozapine and risperidone group according to the accuracy rate of facial emotion recognition expressions (p > .05 for each). At baseline phase, the patients were significantly impaired in recognizing disgusted faces in risperidone than those in clozapine group (p = .032) and it was significantly poorer after treatment with risperidone than with clozapine (p = .031). The patients responded significantly faster after the treatment to all facial emotions except for fearful faces (p = .355). Conclusions: Clozapine and risperidone were not found to have extensive effects on the ability to recognize facial emotions because of ineffectiveness to negative symptoms as in our study. We speculated that the higher dopaminergic receptor occupancy rate of risperidone in insular cortex than that of clozapine might be related with hypo-activation of insula that was associated with particular deficit in ability to recognize expressions of disgust in patients with schizophrenia. Impaired facial emotion recognition ability is present even in first-episode psychosis, which might be a trait marker in schizophrenia

Level of serum thioredoxin and correlation with neurocognitive functions in patients with schizophrenia using clozapine and other atypical antipsychotics

Thioredoxin is a serum antioxidant that has been investigated in the etiology of schizophrenia. The aim of this study is investigating the relationship between serum thioredoxin levels and cognitive functions in acute psychotic episode and remission state patients with schizophrenia; and examining whether there were differences between patients using clozapine and other atypical antipsychotics; including risperidone, olanzapine and amisulpride. This research was performed in schizophrenia patients hospitalized with acute psychotic episode (n=57), reevaluated patients after the initiation of treatment (mean 16 weeks) (n=46), and healthy controls (n=41). Positive and Negative Syndrome Scale, Clinic Global Impressions Scale, Neuropsychologic test battery to assess cognitive performance, and serum thioredoxin levels measured by ELISA were used in this research. Serum thioredoxin levels were highest in acute psychotic episode, lower in the remission state and the lowest in healthy controls. Significant correlation has been established between serum thioredoxin levels and Trail Making Test-A performance in remission state patients. In conclusion, serum thioredoxin levels were increased in acute psychotic episode and decreased in remission state, and its relationship with attention is worth to consider in schizophrenia patients

The impact of cognitive impairment, neurological soft signs and subdepressive symptoms on functional outcome in bipolar disorder

Background: Cognitive impairments and subsyndromal depressive symptoms are present during euthymic periods of bipolar disorder (BD). Most studies have determined that cognitive impairments and residual depressive symptoms have major impacts on psychosocial functioning. The aim of the present study was to identify the major factor responsible for low psychosocial functioning in a subgroup of patients with BD despite clinical recovery

Neuropsychological performance in obsessive-compulsive disorder: A comparison with bipolar disorder and healthy controls

This study examined whether patients with obsessive-compulsive disorder (OCD) have deficits in executive functioning and memory, as well as the specificity of any OCD-related neuropsychological dysfunction. Previous studies have indicated poorer performance among individuals with OCD compared to healthy controls across the majority of neuropsychological domains, however, findings are very inconsistent. We included 34 individuals with bipolar-I disorder (BP-I), 35 untreated patients with OCD, and 33 healthy controls matched for age, gender, and education. Participants completed the Rey Auditory Verbal Learning Test (RAVLT), the Wechsler Memory Scale-Revised (WMS-R) visual-reproduction subscale, and Stroop Color-Word Interference Test (SCWIT). Compared to both healthy controls and participants with OCD, patients with BP-I showed poorer performance in long-delay verbal recall. Although participants with OCD performed more poorly in visual recall than both BP-I patients and healthy controls, their scores were within the normative range. In pairwise comparisons, OCD did not differ from either BP-I or controls. No significant differences were found in verbal memory or Stroop performance between OCD and healthy controls. Overall, we found no significant differences in neuropsychological performance between patients with OCD and healthy controls that could potentially contribute to functional impairment. (C) 2015 Elsevier Inc. All rights reserved

Affective temperaments in subjects with female-to-male gender dysphoria

Background: Males and females have different temperaments. In individuals with gender dysphoria (GD) there is marked incongruence between a person's expressed/experienced gender and their biological sex. The present study aimed to investigate the most Common affective temperaments in individuals with Female-to-male (FtM) GD

The effect of acute organophosphate intoxication on female rat hippocampus cornu ammonis region pyramidal neuron numbers, biochemistry and morphology

WOS: 000486357600008PubMed: 31152871Background: the most commonly used insecticides and pesticides worldwide are organophosphate compounds, chemicals that irreversibly inhibit the cholinesterase enzyme. Acute intoxication with cholinesterase inhibitors is known to cause permanent effects on both the human and rat brains. Aim: To investigate the effect of acute organophosphate intoxication on hippocampus morphology, biochemistry, and pyramidal neuron numbers in female rats. Methods: Twenty-one rats were randomly divided into three groups. the control group received normal nutrition and underwent no procedures. the sham group received intraperitoneal physiological serum, while the experimental group received intraperitoneal 0.8 g/kg fenthion. Rats were sacrificed 24 h after these procedures. the brains were removed and divided in two halves medially, with one side being kept in 10% neutral formalin. After fixation procedures, tissues were embedded in blocks, sliced, and stained. A neuron count was then performed for the hippocampus. the other hippocampus was homogenized and used for biochemical procedures. Results: Hippocampus sections from rats in the experimental group exhibited swelling and loss of shape in pyramidal cells, while no changes were observed in the control or sham groups. the number of neurons in the experimental group was lower than in the control and sham groups. Biochemical analysis revealed higher MDA and GSH values in the experimental group compared to the control and sham groups. Conclusion: Our results show increased apoptotic neurodegeneration of cells in the cornu ammonis region of the hippocampus and changes in biochemical values in rats with acute organophosphate exposure.Ordu University Scientific Research Coordination Office [AP 1703]This study was supported by the Ordu University Scientific Research Coordination Office under project number AP 1703