A Link between Mitochondrial Dysregulation and Idiopathic Autism Spectrum Disorder (ASD): Alterations in Mitochondrial Respiratory Capacity and Membrane Potential

Autism spectrum disorder (ASD) is a neurological disorder triggered by various factors through complex mechanisms. Research has been done to elucidate the potential etiologic mechanisms in ASD, but no single cause has been confirmed. The involvement of oxidative stress is correlated with ASD and possibly affects mitochondrial function. This study aimed to elucidate the link between mitochondrial dysregulation and idiopathic ASD by focusing on mitochondrial respiratory capacity and membrane potential. Our findings showed that mitochondrial function in the energy metabolism pathway was significantly dysregulated in a lymphoblastoid cell line (LCL) derived from an autistic child (ALCL). Respiratory capacities of oxidative phosphorylation (OXPHOS), electron transfer of the Complex I and Complex II linked pathways, membrane potential, and Complex IV activity of the ALCL were analyzed and compared with control cell lines derived from a developmentally normal non-autistic sibling (NALCL). All experiments were performed using high-resolution respirometry. Respiratory capacities of OXPHOS, electron transfer of the Complex I- and Complex II-linked pathways, and Complex IV activity of the ALCL were significantly higher compared to healthy controls. Mitochondrial membrane potential was also significantly higher, measured in the Complex II-linked pathway during LEAK respiration and OXPHOS. These results indicate the abnormalities in mitochondrial respiratory control linking mitochondrial function with autism. Correlating mitochondrial dysfunction and autism is important for a better understanding of ASD pathogenesis in order to produce effective interventions.

Retromer Complex and PI3K Complex II-Related Genes Mediate the Yeast (Saccharomyces cerevisiae) Sodium Metabisulfite Resistance Response

Sodium metabisulfite (Na2S2O5) is widely used as a preservative in the food and wine industry. However, it causes varying degrees of cellular damage to organisms. In order to improve our knowledge regarding its cyto-toxicity, a genome-wide screen using the yeast single deletion collection was performed. Additionally, a total of 162 Na2S2O5-sensitive strains and 16 Na2S2O5-tolerant strains were identified. Among the 162 Na2S2O5 tolerance-related genes, the retromer complex was the top enriched cellular component. Further analysis demonstrated that retromer complex deletion leads to increased sensitivity to Na2S2O5, and that Na2S2O5 can induce mislocalization of retromer complex proteins. Notably, phosphatidylinositol 3-monophosphate kinase (PI3K) complex II, which is important for retromer recruitment to the endosome, might be a potential regulator mediating retromer localization and the yeast Na2S2O5 tolerance response. Na2S2O5 can decrease the protein expressions of Vps34, which is the component of PI3K complex. Therefore, Na2S2O5-mediated retromer redistribution might be caused by the effects of decreased Vps34 expression levels. Moreover, both pharmaceutical inhibition of Vps34 functions and deletions of PI3K complex II-related genes affect cell tolerance to Na2S2O5. The results of our study provide a global picture of cellular components required for Na2S2O5 tolerance and advance our understanding concerning Na2S2O5-induced cytotoxicity effects.

Structure of the Active Nanocomplex of Antiviral and Anti-Infectious Iodine-Containing Drug FS-1

Chromatographic analysis shows that the ionic nanostructured complex of the FS-1 drug contains nanocomplexes of α-dextrin with a size of ~40–48 Å. Based on good agreement between the UV spectra of the model structures and the experimental spectrum of the FS-1 drug, the structure of the active FS-1 nanocomplex is proposed. The structure of the active centers of the drug in the dextrin ring was calculated using the quantum-chemical approach DFT/B3PW91. The active centers, i.e., a complex of molecular iodine with lithium halide (I), a binuclear complex of magnesium and lithium containing molecular iodine, triiodide (II), and triiodide (III), are located inside the dextrin helix. The polypeptide outside the dextrin helix forms a hydrogen bond with dextrin in Complex I and coordinates the molecular iodine in Complex II. It is revealed that the active centers of the FS-1drug can be segregated from the dextrin helix and form complexes with DNA nucleotide triplets. The active centers of the FS-1 drug are only segregated on specific sections of DNA. The formation of a complex between the DNA nucleotide and the active center of FS-1 is a key stage in the mechanisms of anti-HIV, anti-coronavirus (Complex I) and antibacterial action (Complex II).

Immune dysregulation in SHARPIN-deficient mice is dependent on CYLD-mediated cell death

SHARPIN, together with RNF31/HOIP and RBCK1/HOIL1, form the linear ubiquitin chain assembly complex (LUBAC) E3 ligase that catalyzes M1-linked polyubiquitination. Mutations in RNF31/HOIP and RBCK/HOIL1 in humans and Sharpin in mice lead to autoinflammation and immunodeficiency, but the mechanism underlying the immune dysregulation remains unclear. We now show that the phenotype of the Sharpincpdm/cpdm mice is dependent on CYLD, a deubiquitinase previously shown to mediate removal of K63-linked polyubiquitin chains. Dermatitis, disrupted splenic architecture, and loss of Peyer's patches in the Sharpincpdm/cpdm mice were fully reversed in Sharpincpdm/cpdm Cyld−/− mice. We observed enhanced association of RIPK1 with the death-signaling Complex II following TNF stimulation in Sharpincpdm/cpdm cells, a finding dependent on CYLD since we observed reversal in Sharpincpdm/cpdm Cyld−/− cells. Enhanced RIPK1 recruitment to Complex II in Sharpincpdm/cpdm cells correlated with impaired phosphorylation of CYLD at serine 418, a modification reported to inhibit its enzymatic activity. The dermatitis in the Sharpincpdm/cpdm mice was also ameliorated by the conditional deletion of Cyld using LysM-cre or Cx3cr1-cre indicating that CYLD-dependent death of myeloid cells is inflammatory. Our studies reveal that under physiological conditions, TNF- and RIPK1-dependent cell death is suppressed by the linear ubiquitin-dependent inhibition of CYLD. The Sharpincpdm/cpdm phenotype illustrates the pathological consequences when CYLD inhibition fails.

SDHB variant type impacts phenotype and malignancy in pheochromocytoma-paraganglioma

BackgroundTraditional genotype-phenotype correlations for the succinate dehydrogenase-complex II (SDH) genes link SDHB variants to thoracic-abdominal pheochromocytoma-paraganglioma (PPGL) and SDHD variants to head and neck paraganglioma (HNPGL). However, in a recent study we found strong and specific genotype-phenotype associations for SDHD variants. In the present study we zoom in on the genotype-phenotype associations of SDHB gene variants, considering the impact of individual gene variants on disease risk and risk of malignancy.MethodsWe analysed two large independent data sets, including a total of 448 patients with PPGL and HNPGL, and studied the association of missense or truncating SDHB variants with tumour incidence, age of onset and malignancy risk using binomial testing and Kaplan-Meier analysis.ResultsCompared with missense variants, truncating SDHB variants were significantly and consistently more common in patients with PPGL, by a 20 percentage point margin. Malignancy was also significantly more common in truncating versus missense variant carriers. No overall differences in age of PPGL onset were noted between carriers of the two variant types, although some individual variants may differ in certain cases. Missense variants were marginally over-represented among patients with HNPGL, but the difference was not statistically significant.ConclusionSDHB truncating variants convey an elevated risk for development of both PPGL and malignancy compared with missense variants. These results further support earlier robust associations between truncating variants and PPGL, and also suggest that the two variant types differ in their impact on complex II function, with PPGL/HNPGL tissues displaying differing sensitivities to changes in complex II function.

Serine and Threonine Phosphorylation Marks Proteins for Degradation By Clpxp

The mitochondrial serine protease, ClpXP, regulates the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is over-expressed in a subset of AML and inhibiting or hyperactivating it kills leukemic cells and stem cells in vitro and in vivo. Yet, it is unknown how the mitochondrial ClpXP recognizes proteins for degradation. In Bacillus subtilis, the bacterial ClpXP homologue recognizes proteins tagged with phospho-arginine for degradation. To determine if phosphorylated amino acids influence mitochondrial ClpXP-mediated protein degradation, we incubated recombinant ClpXP with its unnatural substrate FITC-casein and increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phospho-tyrosine (pTyr) in a cell-free assay and measured release of fluorogenic FITC. In a dose-dependent manner, pSer and pThr free amino acids inhibited casein cleavage by ClpXP while pTyr, pArg, and the dephosphorylated amino acids had no effect on ClpXP activity. Likewise, ApSA, RRApSVA, and ApTA peptides inhibited ClpXP enzyme activity, while the non-phosphorylated version (ASA, RRASVA, and ATA) had no effect. Next, we tested whether the phosphorylation state of full length proteins would influence their degradation by ClpXP. Using gel-based cell-free assays, the phosphorylation enriched α-casein and β-casein were degraded by recombinant ClpXP. In contrast, κ-casein with low levels of phosphorylation and dephosphorylated α-casein were not cleaved by ClpXP. As ClpX is an AAA ATPase, we asked if pSer and pThr acted on the ATPase of the enzyme. pSer and pThr did not inhibit the ATPase activity of ClpX. We also measured the effect of pSer and pThr on the peptidase activity of ClpP alone without its regulatory subunit ClpX. Neither pSer or pThr inhibited ClpP peptidase activity. We investigated if pSer and pThr could bind to ClpX. Using thermal shift binding assays, we demonstrated that pSer and pThr but not pTyr and pArg bound ClpX and none of the phosphorylated amino acids bound ClpP. Similarly, ApSA, RRApSVA, and ApTA peptides bound ClpX, while the non-phosphorylated ASA, RRASVA, and ATA did not bind ClpX. Previously we showed that ClpP interacted with respiratory chain complex II subunit SDHA, and ClpP knockdown in AML cells impaired respiratory chain complex II activity and reactive oxygen species increased. Therefore, we tested how ClpXP knockdown impacts levels of phospho-serine SDHA. Using shRNA, we knocked down ClpP and ClpX individually in OCI-AML2 cells. After target knockdown, we pulled down pSer proteins and then probed for SDHA. Knockdown of both ClpP and ClpX increased the abundance of serine phosphorylated SDHA comparing to control, suggesting that ClpXP degrades serine phosphorylated SDHA. Finally, as a chemical approach, we generated small molecules that mimic pSer and demonstrated that they inhibited ClpXP-mediated degradation of FITC-casein and bound ClpX with a potency similar to pSer. In summary, we discovered that ClpX binds pSer and pThr and phosphorylation of these amino acids mark proteins for degradation by the ClpXP mitochondrial protease. This work highlights a new strategy to develop inhibitors of ClpXP for the treatment of AML. Disclosures Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; UHN: Patents & Royalties.