Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • Vacuolin-1 In order to determine which complex of

    2022-02-09

    In order to determine which complex of the electron transport chain is targeted by Authipyrin, a semi-intact assay was performed. The substrates of each complex were added separately in combination with Authipyrin or the respective control inhibitor. Seahorse XF plasma membrane permeabilizer (PMP) was used to ensure substrate availability. Authipyrin selectively inhibited complex I (A), but had no effect on the activity of complex II to IV (B–D). These results are in line with previously published data on the autophagy inhibitor Aumitin. Aumitin also targets mitochondrial complex I and has an inhibitory effect on autophagy. Furthermore, the complex I inhibitor Rotenone was identified as a modulator of autophagy. Authipyrin could cause complex I inhibition by two different mechanisms. On the one hand, the Vacuolin-1 could directly target complex I to interfere with mitochondrial respiration. On the other hand Authipyrin could target a protein involved in NADH generation and thus interfere with the NADH supply chain. This would result in complex I substrate depletion and consequently decrease of oxidative phosphorylation. To investigate, whether the inhibitory effect of Authipyrin on complex I is direct or indirect, an assay with isolated bovine heart mitochondria was performed. This assay showed that Authipyrin dose-dependently impaired NADH-Coenyme Q reductase activity (E). This assay requires higher concentrations of Authipyrin in comparison to the semi-intact assay in order to achieve complex I inhibition. This is presumably due to the fact that isolated bovine heart mitochondria were employed, which results in a higher concentration of complexI than under cellular conditions. Similar results were also observed for the complex I inhibitor Aumitin. Conclusively, this assay shows that Authipyrinis a direct inhibitor of complex I (E). In conclusion, we demonstrated that the thienopyrimidine-containing molecule Authipyrin is a highly potent inhibitor of starvation, as well as rapamycin induced autophagy. Thus, it is likely that Authipyrinacts downstream of mTOR. Although thienopyrimidine is a known kinase inhibitory scaffold, autophagy modulation of Authipyrin is not connected to a kinase target. We demonstrated that Authipyrin downregulates mitochondrial respiration by affecting mitochondrial complex I. An assay with isolated bovine heart mitochondria showed that Authipyrinhas a direct effect on NADH-CoQ reductase activity. Alterations of mitochondrial metabolism and complex I activity are known to influence autophagic flux. Furthermore, dysfunctional mitochondria and impaired autophagy are characteristic for Parkinson’s disease. However, the exact mechanism underlying the interplay between autophagic flux and mitochondrial respiration remains to be elucidated. Thus, structurally novel tool compounds, such as Authipyrin are highly valuable for investigation of this interdependency and can facilitate the development of more efficient therapeutics.
    Principles of Protein Kinase Substrate Specificity Protein kinases selectively target specific substrates through several types of physical interactions (Figure 1) 1, 2. For example, it is self-evident that the phosphorylated amino acid residue must interact at least transiently with the active site of the kinase. Eukaryotic protein kinases are generally subdivided into tyrosine kinases (TyrKs), serine–threonine kinases (STKs), and dual-specificity kinases based on their favored substrate phosphoacceptor residues, which are determined by conserved features of the kinase active site unique to each class [3]. As with other protein-modifying enzymes, kinases have broad catalytic clefts that accommodate multiple residues flanking the site of phosphorylation, leading to specificity at the level of phosphorylation site sequence 4, 5. However, as a rule, catalytic site interactions alone are insufficient to mediate selection of protein substrates. Kinase recognition motifs typically consist of only one to three residues that are critical for efficient phosphorylation (Table 1). As a consequence, essentially all proteins will harbor sites matching the simplest of these motifs, and there will be thousands of occurrences of more stringent motifs within a proteome. Furthermore, related kinases can have identical phosphorylation site motifs, yet mediate different cellular functions through phosphorylation of distinct substrates [6]. Phosphorylation site interactions may therefore be primarily important in determining which specific residues within a substrate protein are phosphorylated, and in cooperating with additional interactions to select substrates.