Gao, T.-Y. | Search bioRxiv

bioRxiv2024

Elevated Ubiquitin Phosphorylation by PINK1 Contributes to Proteasomal Impairment and Promotes Neurodegeneration

Ubiquitin (Ub), a central regulator of protein turnover, can be phosphorylated by PINK1 (PTEN-induced putative kinase 1) to generate S65-phosphorylated ubiquitin (pUb). Elevated pUb levels have been observed in aged human brains and in Parkinsons disease, but the mechanistic link between pUb elevation and neurodegeneration remains unclear. Here, we demonstrate that pUb elevation is a common feature under neurodegenerative conditions, including Alzheimers disease, aging, and ischemic injury. We show that impaired proteasomal activity leads to the accumulation of sPINK1, the cytosolic form of PINK1 that is normally proteasome-degraded rapidly. This accumulation increases ubiquitin phosphorylation, which then inhibits ubiquitin-dependent proteasomal activity by interfering with both ubiquitin chain elongation and proteasome-substrate interactions. Specific expression of sPINK1 in mouse hippocampal neurons induced progressive pUb accumulation, accompanied by protein aggregation, proteostasis disruption, neuronal injury, neuroinflammation, and cognitive decline. Conversely, pink1 knockout mitigated protein aggregation in both mouse brains and HEK293 cells. Furthermore, the detrimental effects of sPINK1 could be counteracted by co-expressing Ub/S65A phospho-null mutant but exacerbated by over-expressing Ub/S65E phospho-mimic mutant. Together, these findings reveal that pUb elevation, triggered by reduced proteasomal activity, inhibits proteasomal activity and forms a feedforward loop that drives progressive neurodegeneration.

biochemistry↗

bioRxiv2024

Explore the mechanism of Zigui Yichong Formula in reducing the apoptosis of ovarian granulosa cells in premature ovarian insufficiency based on network pharmacology, molecular docking and cell experiments

ObjectiveThis research is conducted with the objective of exploring the underlying mechanism by which the Zigui Yichong Formula (ZGYCF) diminishes granulosa cell apoptosis in the context of premature ovarian insufficiency (POI), utilizing network pharmacology, molecular docking, and cellular experimentation approaches. MethodsThe active constituents and potential therapeutic targets of the 12 medicinal herbs in ZGYCF, which include Rehmannia glutinosa, Cervus nippon, Cornus officinalis, Ligustrum lucidum, Lycium barbarum, Paeonia lactiflora, Astragalus membranaceus, Codonopsis pilosula, Atractylodes macrocephala, Angelica sinensis, Cyperus rotundus, and Glycyrrhiza uralensis, were identified through searches in the TCMSP, BATMAN, HERB, and ETCM databases. Targets associated with the POI condition were gathered from the OpenTargets, DrugBank, and GeneCards databases. Subsequently, a Venn diagram illustrating the compound-target-disease interaction was generated to derive a set of common targets that bridge the gap between pharmacological and pathological targets. A drug-component-target-disease network diagram was created using Cytoscape 3.9.1. Additionally, protein-protein interaction (PPI) networks were built utilizing the STRING database and visualized with Cytoscape to pinpoint key targets within the overlapping target set. Functional annotation and pathway enrichment analyses, including GO and KEGG pathway analyses, were performed using the clusterProfiler package in R 4.2.1 to investigate the underlying mechanisms by which the drug may influence the disease state. The molecular docking of pivotal active constituents with central targets was carried out using AutoDock Tools. Following this, in vitro studies were executed to corroborate the anticipated mechanisms of action of ZGYCF on POI that were inferred from the network pharmacology analysis. ResultsThe selected active components include quercetin, kaempferol, and {beta}-sitosterol. The core targets identified are Tp53, Bcl-2, and Caspase-3. GO functional and KEGG enrichment analyses indicate that these core targets are primarily enriched in the p53 signaling pathway. Molecular docking results show that quercetin, kaempferol, and {beta}-sitosterol have good binding affinity with TP53, Bcl-2, and Caspase-3. Additionally, in vitro experiments demonstrate that ZGYCF medicated serum can reduce ACR-induced apoptosis in KGN cells, increase Bcl-2 expression, and decrease the expression of p53, Bax, Caspase-3, and the Bcl-2/Bax ratio. ConclusionZGYCF exerts therapeutic effects on POI through multiple targets and pathways, and it may reduce ACR-induced apoptosis in KGN cells by modulating the p53 signaling pathway. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=130 HEIGHT=200 SRC="FIGDIR/small/614279v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1e55e2forg.highwire.dtl.DTLVardef@18a67d0org.highwire.dtl.DTLVardef@1a596d0org.highwire.dtl.DTLVardef@503fde_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

Ubiquitin phosphorylation accelerates protein aggregation and promotes neurodegeneration in the aging brain

Ser65-phosphorylated ubiquitin (pUb) was found elevated in neurons of aged and neurodegenerative brains. Yet little is known whether a causative link exists between pUb level and brain aging. Here we show that the knockout of pink1, a Ub kinase, abolished pUb elevation and decelerated protein aggregation in aged mouse brains and cells with proteasomal inhibition. Conversely, over-expression of PINK1 but not the kinase-dead version increased the pUb level and accelerated protein aggregation by suppressing of proteasomal degradation. Furthermore, PINK1 over-expression in mouse hippocampus neurons increased pUb level and protein aggregation, slowly leading to mitochondrial injury, neurodegeneration, and cognitive impairment. Notably, the neuronal damages induced by PINK1 were rescued by the dominant negative Ub/S65A mutant, while Ub/S65E phosphomimetic mutant caused neuronal death. Together, an incidental increase of Ub phosphorylation can progressively and cumulatively cause the decline of Ub-dependent proteasomal activity, consequenting promotes neurodegeneration in the aging brain.

cell biology↗