The contribution of proteasomal impairment to autophagy activation by C9orf72 poly-GA aggregates.

BACKGROUND: Poly-GA, a dipeptide repeat protein unconventionally translated from GGGGCC (G4C2) repeat expansions in C9orf72, is abundant in C9orf72-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9orf72-ALS/FTD). Although the poly-GA aggregates have been identified in C9orf72-ALS/FTD neurons, the effects on UPS (ubiquitin-proteasome system) and autophagy and their exact molecular mechanisms have not been fully elucidated. RESULTS: Herein, our in vivo experiments indicate that the mice expressing ploy-GA with 150 repeats instead of 30 repeats exhibit significant aggregates in cells. Mice expressing 150 repeats ploy-GA shows behavioral deficits and activates autophagy in the brain. In vitro findings suggest that the poly-GA aggregates influence proteasomal by directly binding proteasome subunit PSMD2. Subsequently, the poly-GA aggregates activate phosphorylation and ubiquitination of p62 to recruit autophagosomes. Ultimately, the poly-GA aggregates lead to compensatory activation of autophagy. In vivo studies further reveal that rapamycin (autophagy activator) treatment significantly improves the degenerative symptoms and alleviates neuronal injury in mice expressing 150 repeats poly-GA. Meanwhile, rapamycin administration to mice expressing 150 repeats poly-GA reduces neuroinflammation and aggregates in the brain. CONCLUSION: In summary, we elucidate the relationship between poly-GA in the proteasome and autophagy: when poly-GA forms complexes with the proteasome, it recruits autophagosomes and affects proteasome function. Our study provides support for further promoting the comprehension of the pathogenesis of C9orf72, which may bring a hint for the exploration of rapamycin for the treatment of ALS/FTD.

GADD45ß stabilized by direct interaction with HSP72 ameliorates insulin resistance and lipid accumulation.

Growth arrest and DNA damage-inducible 45ß (GADD45ß) belongs to the GADD45 family which is small acidic proteins in response to cellular stress. GADD45ß has already been reported to have excellent capabilities against cancer, innate immunity and neurological diseases. However, there is little information regard GADD45ß and non-alcoholic fatty liver disease (NAFLD). In the current work, we found that the expression of GADD45ß was markedly decreased in the livers of NAFLD patients via analyzing Gene Expression Omnibus (GEO) dataset and in mouse model through detecting its mRNA in high-fat-high-fructose diet (HFHFr)-fed mice. Moreover, the results from in vivo experiment demonstrated that overexpression of GADD45ß by AAV8-mediated gene transfer in HFHFr-fed mouse model could reduce the level of serum and hepatic triglyceride (TG), and alleviate insulin resistance. Subsequently, by combining immunoprecipitation (IP) and mass spectrometry, we identified that HSP72 directly interacted with GADD45ß to prevent GADD45ß from being degraded by the proteasome pathway. Finally, the benefits of GADD45ß in regulating key factors of TG synthesis and insulin signaling pathway were abolished after HSP72 knockdown. In conclusion, GADD45ß stabilized by the interaction with HSP72 could alleviate the NAFLD-related pathologies, suggested it might be a potential target for the treatment of NAFLD.

miR-34a-5p regulates PINK1-mediated mitophagy via multiple modes.

AIMS: PTEN induced putative kinase 1 (PINK1)-mediated mitophagy process is tightly associated with various age-dependent diseases in mammals. The roles of miRNAs (miRNAs) in the PINK1-mediated mitophagy process are not fully understood. Here we discovered that miR-34a-5p suppresses PINK1 expression directly though two post-transcriptional non-classical binding modes, resulting in inhibition of PINK1-mediated mitophagy process. MAIN METHODS: For in vivo experiments, brains were dissected from 8 weeks old and 40 weeks old C57BL/6 male mice to measure miR-34a-5p expression and PINK1 expression. For in vitro experiments, overexpression of miR-34a-5p mimics in HEK293 cells was performed to investigate the effect of miR-34a-5p on PINK1 expression and its regulatory mechanism, parkin recruitment and mitophagy process. KEY FINDINGS: The level of miR-34a-5p was upregulated and the level of PINK1 mRNA was downregulated in brains of aged mice. Both the 3'-untranslated region (3'UTR) and the Coding DNA sequence (CDS) of PINK1 mRNA were bound to the non-seed region of miR-34a-5p, rather than the seed region, resulting in a decrease in PINK1 expression. Endogenous miR-34a-5p knockout increased PINK1 expression. Further results indicated that miR-34a-5p inhibits mitophagy process by reduction of PINK1. miR-34a-5p hinders phosphorylated Ser65-ubiquitin (pS65-Ub) accumulation, prevents the mitochondrial recruitment of Parkin, attenuates ubiquitination and delays the clearance of damaged mitochondria. SIGNIFICANCE: We firstly found that miR-34a-5p suppresses PINK1 directly and further regulates mitophagy through non-canonical modes. This finding hints at a crucial role of miR-34a-5p implicated in accelerating the pathogenesis of age-related neurological diseases.

Antigenic activity and epitope analysis of ß-conglycinin hydrolyzed by pepsin.

BACKGROUND: Soybean is among the 'big eight' allergenic foods, and ß-conglycinin, the main antigenic protein of soybean, has high levels of antigenic activity. Why the antigenic activity of soybean ß-conglycinin is not eliminated by enzymatic hydrolysis is not clear. In this study, changes in the molecular composition and antigenicity of ß-conglycinin hydrolyzed by pepsin were analyzed and it was determined whether complete sequential epitopes exist in the resulting hydrolysates. The nature and antigenic activity of protein subunits obtained after ß-conglycinin hydrolysis were also assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and competitive enzyme-linked immunosorbent assay, respectively. RESULTS: The residual antigenic activity of ß-conglycinin was 52%, α'- and α-subunits completely disappeared, the 49 kDa fraction partially disappeared, and peptides measuring 27 and 23 kDa were newly formed after 60 min of enzymatic hydrolysis. Prolonged enzymatic hydrolysis did not result in remarkable changes in these peptides; thus, the peptides show some resistance to enzymatic hydrolysis. The amino acid sequences of the peptide chains were analyzed by matrix-assisted laser desorption / ionization-time of flight mass spectrometry and aligned with the related sequences in the corresponding protein and antigen databases. Ten complete sequential epitopes were identified in the residual 49 kDa fraction, of these epitopes, two were from α-subunits and eight were from ß-subunits. CONCLUSION: The presence of complete sequential epitopes in hydrolysates obtained from the enzymatic hydrolysis of soybean is an important reason for the incomplete disappearance of the antigenic activity of ß-conglycinin. © 2020 Society of Chemical Industry.

Preparation of mesoporous and/or macroporous SnO2-based powders and their gas-sensing properties as thick film sensors.

Mesoporous and/or macroporous SnO(2)-based powders have been prepared and their gas-sensing properties as thick film sensors towards H(2) and NO(2) have been investigated. The mesopores and macropores of various SnO(2)-based powders were controlled by self-assembly of sodium bis(2-ethylhexyl)sulfosuccinate and polymethyl-methacrylate (PMMA) microspheres (ca. 800 nm in diameter), respectively. The introduction of mesopores and macropores into SnO(2)-based sensors increased their sensor resistance in air significantly. The additions of SiO(2) and Sb(2)O(5) into mesoporous and/or macroporous SnO(2) were found to improve the sensing properties of the sensors. The addition of SiO(2) into mesoporous and/or macroporous SnO(2) was found to increase the sensor resistance in air, whereas doping of Sb(2)O(5) into mesoporous and/or macroporous SnO(2) was found to markedly reduce the sensor resistance in air, and to increase the response to 1,000 ppm H(2) as well as 1 ppm NO(2) in air. Among all the sensors tested, meso-macroporous SnO(2) added with 1 wt% SiO(2) and 5 wt% Sb(2)O(5), which were prepared with the above two templates simultaneously, exhibited the largest H(2) and NO(2) responses.