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Early and precise diagnosis of a-synucleinopathies is challenging but critical. In this study, we developed a molecular beacon-based assay to evaluate microRNA-containing extracellular vesicles (EVs) in plasma. We recruited 1203 participants including healthy controls (HCs) and patients with isolated REM sleep behavior disorder (iRBD), a-synucleinopathies, or non-a-synucleinopathies from eight centers across China. Plasma miR-44438-containing EV levels were significantly increased in a-synucleinopathies, including those in the prodromal stage (e.g., iRBD), compared to both non-a-synucleinopathy patients and HCs. However, there are no significant differences between Parkinson's disease (PD) and multiple system atrophy. The miR-44438-containing EV levels negatively correlated with age and the Hoehn and Yahr stage of PD patients, suggesting a potential association with disease progression. Furthermore, a longitudinal analysis over 16.3 months demonstrated a significant decline in miR-44438-containing EV levels in patients with PD. These results highlight the potential of plasma miR-44438-containing EV as a biomarker for early detection and progress monitoring of a-synucleinopathies. [ABSTRACT FROM AUTHOR]

Mutations in several genes, including PINK1 and Parkin, are known to cause autosomal recessive cases of Parkinsons disease in humans. These genes operate in the same pathway and play a crucial role in mitochondrial dynamics and maintenance. PINK1 is required to recruit Parkin to mitochondria and initiate mitophagy upon mitochondrial depolarization. In this study, we show that PINK1-dependent Parkin mitochondrial recruitment in response to global mitochondrial damage by carbonyl cyanide m-chlorophenyl hydrazine (CCCP) requires active glucose metabolism. Parkin accumulation on mitochondria and subsequent Parkin-dependent mitophagy is abrogated in glucose-free media or in the presence of 2-deoxyglucose upon CCCP treatment. The defects in Parkin recruitment correlate with intracellular ATP levels and can be attributed to suppression of PINK1 up-regulation in response to mitochondria depolarization. Low levels of ATP appear to prevent PINK1 translation instead of affecting PINK1 mRNA expression or reducing its stability. Consistent with a requirement of ATP for elevated PINK1 levels and Parkin mitochondrial recruitment, local or individual mitochondrial damage via photoirradiation does not affect Parkin recruitment to damaged mitochondria as long as a pool of functional mitochondria is present in the photoirradiated cells even in glucose-free or 2-DG-treated conditions. Thus, our data identifies ATP as a key regulator for Parkin mitochondrial translocation and sustaining elevated PINK1 levels during mitophagy. PINK1 functions as an AND-gate and a metabolic sensor coupling biogenetics of cells and stress signals to mitochondria dynamics. [ABSTRACT FROM AUTHOR]

p27Kip1 is an essential cell cycle inhibitor of Cyclin-dependent kinases. Ubiquitin-mediated proteolysis of p27Kip1 is an important mechanism for activation of Cyclin E-Cdk2 and facilitates G1/S transition. Ubiquitination of p27 is primarily catalyzed by a multisubunit E3 ubiquitin ligase, SCFSkp2, and requires an adapter protein Cks1. In addition, phosphorylation of p27 at Thr187 by Cyclin E and Cdk2 is also essential for triggering substrate ubiquitination. Here we investigate the molecular mechanism of p27 ubiquitination. We show that Cyclin E-Cdk2 is essential for targeting the p27 substrate to SCFSkp2. Direct physical contact between Cyclin E but not Cdk2 and p27 is required for p27 recruitment to SCFSkp2. In a search for positively charged amino acid residues that may be involved in recognition of the Thr187 phosphate group, we found that Arg306 of Skp2 is required for association and ubiquitination of phosphorylated p27 but dispensable for ubiquitination of unphosphorylated p21. Thus, our data unravel the molecular organization of the ubiquitination complex that catalyzes p27 ubiquitination and provide unique insights into the specificity of substrate recognition by SCFSkp2. [ABSTRACT FROM AUTHOR]

A critical function of the PTEN-induced kinase 1 (PINK1)- Parkin pathway is to mediate the clearing of unhealthy or damaged mitochondria via mitophagy. Loss of either PINK1 or Parkin protein expression is associated with Parkinson's disease. Here, using a high-throughput screening approach along with recombinant protein expression and kinase, immunoblotting, and immunofluorescence live-cell imaging assays, we report that celastrol, a pentacyclic triterpenoid isolated from extracts of the medicinal plant Tripterygium wilfordii, blocks recruitment pof Parkin to mitochondria, preventing mitophagy in response to mitochondrial depolarization induced by carbonyl cyanide m-chlorophenylhydrazone or to gamitrinib-induced inhibition of mitochondrial heat shock protein 90 (HSP90). Celastrol's effect on mitophagy was independent of its known role in microtubule disruption. Instead, we show that celastrol suppresses Parkin recruitment by inactivating PINK1 and preventing it from phosphorylating Parkin and also ubiquitin. We also observed that PINK1 directly and strongly associates with TOM20, a component of the translocase of outer mitochondrial membrane (TOM) machinery and relatively weak binding to another TOM subunit, TOM70. Moreover, celastrol disrupted binding between PINK1 and TOM20 both in vitro and in vivo but did not affect binding between TOM20 and TOM70. Using native gel analysis, we also show that celastrol disrupts PINK1 complex formation upon mitochondrial depolarization and sequesters PINK1 to high-molecular-weight protein aggregates. These results reveal that celastrol regulates the mitochondrial quality control pathway by interfering with PINK1-TOM20 binding. [ABSTRACT FROM AUTHOR]

Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m-chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib. [ABSTRACT FROM AUTHOR]

Mpsl is a protein kinase that regulates normal mitotic pro- gression and the spindle checkpoint in response to spindle damage. The levels of Mpsl are relatively low in cells during interphase but elevated in mitosis or upon activation of the spindle checkpoint, although the dynamic range of Mpsl ex- pression and the Mpsl catalytic mechanism have not been carefully characterized. Our recent structural studies of the Mpsl kinase domain revealed that the carboxyl-terminal tail region of Mpsl is unstructured, raising the question of whether this region has any functional role in Mpsl catalysis. Here we first determined the cellular abundance of Mpsl dur- ing cell cycle progression and found that Mpsl levels vary be- tween 60,000 per cell in early G1 and 110,000 per cell during mitosis. We studied phosphorylation of a number of Mpsl substrates in vitro and in culture cells. Unexpectedly, we found that the unstructured carboxyl-terminal region of Mpsl plays an essential role in substrate recruitment. Kinetics studies us- ing the purified recombinant wild type and mutant kinases indicate that the carboxyl-terminal tail is largely dispensable for autophosphorylation of Mpsl but critical for trans-phos- phorylation of substrates in vitro and in cultured cells. Mpsl mutant without the unstructured tail region is defective in me diating spindle assembly checkpoint activation. Our results underscore the importance of the unstructured tail region of Mpsl in kinase activation. [ABSTRACT FROM AUTHOR]

The primary intracellular mediators of TGF-β signaling are the Smad proteins. Phosphorylation of R-Smad at the C-terminal SSXS motif by the activated TGF-β type I receptor kinase triggers a conformation change in R-Smad and facilitates complex formation between R-Smad and Smad4, which shuttle into the nucleus where they interact with DNA and other transcription factors to regulate gene expression. In an attempt to identify proteins interacting with activated Smad signaling complex, we discovered that Mps1, a protein kinase that plays important roles in normal mitotic progression and mitotic checkpoint signaling, co-purifies with this complex. We demonstrated that Smad2 and Smad3 but not Smad4 are substrates of Mps1 in vitro and in vivo. Mps1 phosphorylates Smad2 and Smad3 at the SSXS motif in their C-terminal regions in vitro and in vivo. Disruption of microtubule networks by nocodazole activates Mps1 and promotes TGF-β-independent activation of Smad signaling. We found that Mps1 is involved in turning on Smad signaling by phosphorylating R-Smads. Our results reveal a novel functional link between Mps1 and Smads in a non-canonical Smad signaling pathway. [ABSTRACT FROM AUTHOR]

The broad range of biological responses elicited by transforming growth factor-β (TGF-β) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. Here we show that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-β (GSK3-β). Smad3 physically interacts with Axin and GSK3-β only in the absence of TGF-β. Reduction in the expression or activity of Axin/GSK3-β leads to increased Smad3 stability and transcriptional activity without affecting TGF-β receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-β. Mechanistically, Axin facilitates GSK3-β-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-β, and suggest a new function of the Axin/GSK3-β complex in modulating critical TGF-β/Smad3-regulated processes during development and tumor progression. [ABSTRACT FROM AUTHOR]

From 2019 to 2020, the Mexican economy declined for two consecutive years, especially in the last one when it was hit by a decline of 8.4% before the COVID-19 pandemic impacts which was not only one of the worst in the OECD club, but also the deepest economic recession since 1932 in the national history. At the same time, both the number of people in poverty and poverty rate in 2020 have increased compared with those registered in 2018. Through the analysis, we can find that the current Mexican government has increased the intensity and scope of the implementation of social relief policies adhered to the principal of "for the good of all, first the poor (Por el bien de todos, Primero los pobres)." However, in the context of recession caused by the COVID-19, neither the general decrease in residents' income could be avoided, nor the number of people in poverty has been reduced. Besides, in accordance with the benefits obtained by the distinct household deciles based on the income and expenditure survey published by INEGI, it showed that the implementation of government relief measures has relatively reduced the support for the low-income people and further aggravated the deterioration of poverty due to its indifferent application with respect to high-income households and the low-income ones. Therefore, the deficiencies in the response implemented in the face of the epidemic, especially poverty alleviation actions and social relief policies, have further enhanced the poverty problem at least partially. In this sense, recover and improve the economic growth rate as soon as possible will not enough to reduce the poverty, and it should be accompanied by the necessary adjustments in the poverty alleviation measures and social relief policies, especially with a focalized approach inclined to the low-income segments of the population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shuying and Xuedong.)