The ciliary protein RPGRIP1L governs autophagy independently of its proteasome-regulating function at the ciliary base in mouse embryonic fibroblasts

<p>Previously, macroautophagy/autophagy was demonstrated to be regulated inter alia by the primary cilium. Mutations in <i>RPGRIP1L</i> cause ciliary dysfunctions resulting in severe human diseases summarized as ciliopathies. Recently, we showed that <i>RPGRIP1L</i> deficiency leads to a decreased proteasomal activity at the ciliary base in mice. Importantly, the drug-induced restoration of proteasomal activity does not rescue ciliary length alterations in the absence of RPGRIP1L indicating that RPGRIP1L affects ciliary function also via other mechanisms. Based on this knowledge, we analyzed autophagy in <i>Rpgrip1l</i>-negative mouse embryos. In these embryos, autophagic activity was decreased due to an increased activation of the MTOR complex 1 (MTORC1). Application of the MTORC1 inhibitor rapamycin rescued dysregulated MTORC1, autophagic activity and cilia length but not proteasomal activity in <i>Rpgrip1l</i>-deficient mouse embryonic fibroblasts demonstrating that RPGRIP1L seems to regulate autophagic and proteasomal activity independently from each other.</p

Systematic analysis of ATG13 domain requirements for autophagy induction

<p>Macroautophagy/autophagy is an evolutionarily conserved cellular process whose induction is regulated by the ULK1 protein kinase complex. The subunit ATG13 functions as an adaptor protein by recruiting ULK1, RB1CC1 and ATG101 to a core ULK1 complex. Furthermore, ATG13 directly binds both phospholipids and members of the Atg8 family. The central involvement of ATG13 in complex formation makes it an attractive target for autophagy regulation. Here, we analyzed known interactions of ATG13 with proteins and lipids for their potential modulation of ULK1 complex formation and autophagy induction. Targeting the ATG101-ATG13 interaction showed the strongest autophagy-inhibitory effect, whereas the inhibition of binding to ULK1 or RB1CC1 had only minor effects, emphasizing that mutations interfering with ULK1 complex assembly do not necessarily result in a blockade of autophagy. Furthermore, inhibition of ATG13 binding to phospholipids or Atg8 proteins had only mild effects on autophagy. Generally, the observed phenotypes were more severe when autophagy was induced by MTORC1/2 inhibition compared to amino acid starvation. Collectively, these data establish the interaction between ATG13 and ATG101 as a promising target in disease-settings where the inhibition of autophagy is desired.</p

Additional file 1 of The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin

Additional file 1: Figure S1. Schematic representation of the thermal proteome profiling temperature range (TPP-TR) workflow. HeLa wt cells were treated with 100 nM prodigiosin or DMSO for 6 h. After the incubation, cells were harvested, washed and aliquots of the cell suspensions were exposed to short (3 min) treatments at different temperatures in the range between 36.5 °C and 67 °C. Cells were lysed and the non-denatured protein fraction was recovered after centrifugation. Quantitative protein analysis was performed by immunoblotting (CETSA) or MS (TPP). For MS, proteins underwent tryptic digest and the resulting peptides were labeled using TMT 10plex. The samples were combined such that prodigiosin treated and corresponding control samples belonging to the same temperature were analyzed within the same TMT set (similarly as described before for RTSA), allowing for studying not only thermal stability but also abundance effects upon prodigiosin treatment

Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade of autophagy

<p>Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the serine/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.</p