1. Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth
Xing Feng, et al. Autophagy. 2019 Jul;15(7):1130-1149. doi: 10.1080/15548627.2019.1570063. Epub 2019 Jan 27.
UVRAG (UV radiation resistance associated) is an important regulator of mammalian macroautophagy/autophagy by interacting with BECN1, PIK3C3, and RUBCN. Phosphorylation of UVRAG by MTORC1 negatively regulates autophagosome maturation under nutrient-enriched conditions. However, how UVRAG ubiquitination is regulated is still unknown. Here we report that UVRAG is ubiquitinated by SMURF1 at lysine residues 517 and 559, which decreases the association of UVRAG with RUBCN and promotes autophagosome maturation. However, the deubiquitinase ZRANB1 specifically cleaves SMURF1-induced K29 and K33-linked polyubiquitin chains from UVRAG, thereby increasing the binding of UVRAG to RUBCN and inhibiting autophagy flux. We also demonstrate that CSNK1A1-mediated UVRAG phosphorylation at Ser522 disrupts the binding of SMURF1 to UVRAG through PPxY motif and blocks UVRAG ubiquitination-mediated autophagosome maturation. Interestingly, ZRANB1 is phosphorylated at Thr35, and Ser209 residues by CSNK1A1, and this phosphorylation activates its deubiquitinating activity. Importantly, we provide in vitro and in vivo evidence that UVRAG ubiquitination at lysine residues 517 and 559 or prevention of Ser522 phosphorylation by D4476, a CSNK1A1 inhibitor, enhances the lysosomal degradation of EGFR, which significantly inhibits hepatocellular carcinoma (HCC) growth. Furthermore, UVRAG S522 phosphorylation levels correlate with ZRANB1 T35/S209 phosphorylation levels and poor prognosis in HCC patients. These findings identify a novel molecular mechanism by which ubiquitination and phosphorylation of UVRAG regulate its function in autophagosome maturation and HCC growth, encouraging further study of their potential therapeutic implications.
2. Identification of an autoinhibitory, mitophagy-inducing peptide derived from the transmembrane domain of USP30
Xuan Qin, Rui Wang, Hongkun Xu, Licheng Tu, Hailing Chen, Heng Li, Na Liu, Jinpeng Wang, Shuiming Li, Feng Yin, Naihan Xu, Zigang Li Autophagy. 2022 Sep;18(9):2178-2197. doi: 10.1080/15548627.2021.2022360. Epub 2022 Jan 6.
The mitochondrial-anchored deubiquitinating enzyme USP30 (ubiquitin specific peptidase 30) antagonizes PRKN/parkin-mediated mitophagy, making it a potential target for treating Parkinson disease. However, few inhibitors targeting USP30 have been reported. Here, we report a novel peptide (Q14) derived from the transmembrane (TM) domain of USP30 that can target mitochondrial-anchored USP30 directly and increase mitophagy through two intriguing and distinct mechanisms: a novel autoinhibition mechanism in USP30 and accelerated autophagosome formation via the LC3-interacting region (LIR) of the Q14 peptide. We identified the potential binding sites between the Q14 peptide and USP30 and postulated that an allosteric autoinhibition mechanism regulates USP30 activity. Furthermore, the LIR motif in the Q14 peptide offers additional binding with LC3 and accelerated autophagosome formation. The two mechanisms synergistically enhance mitophagy. Our work provides novel insight and direction to the design of inhibitors for USP30 or other deubiquitinating enzymes (DUBs).
3. Enzyme-sensing chitosan hydrogels
Mir Morteza Sadat Ebrahimi, Holger Schönherr Langmuir. 2014 Jul 8;30(26):7842-50. doi: 10.1021/la501482u. Epub 2014 Jun 24.
We report on a chitosan hydrogel-based platform for the detection of enzymes, which is compatible with the implementation in infection-sensing wound dressings. Thin films of the established wound dressing biopolymer chitosan were functionalized with a fluorogenic substrate, which is released upon enzymatic degradation, resulting in a pronounced increase in fluorescence emission intensity. In this first model study, the fluorogenic substrate alanyl-alanyl-phenylalanine-7-amido-4-methylcoumarin (AAP-AMC) was covalently conjugated via amide bond formation to chitosan and was shown to facilitate the detection of the serine protease α-chymotrypsin. Systematic investigations established the dependence of hydrogel thickness and substrate loading on the hydrogel preparation conditions, as well as the dependence of the rate of the reaction on the initial enzyme concentration and the loading of AAP-AMC in the hydrogel. The initial release rate of the fluorophore 7-AMC was found to be linear with enzyme concentration and substrate loading and was independent of hydrogel thickness. Under optimized conditions the hydrogel reports the presence of α-chymotrypsin in <5 min with a limit of detection of ≤10 nM. This generic approach, which can be adapted to detect different kinds of enzymes by using appropriate fluorogenic or chromogenic substrates, is highly interesting for targeting the detection of specific pathogenic bacteria, e.g., in wound dressings.
