bims-apauto 2021-10-24 papers

Issue of 2021‒10‒24
six papers selected by
Su Hyun Lee
Seoul National University

Proc Natl Acad Sci U S A. 2021 Oct 26. pii: e2115430118. [Epub ahead of print]118(43):

Aminopeptidases trim Xaa-Pro proteins, initiating their degradation by the Pro/N-degron pathway.

Shun-Jia Chen, Leehyeon Kim, Hyun Kyu Song, Alexander Varshavsky.

N-degron pathways are proteolytic systems that recognize proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Our previous work identified Gid4 as a recognition component (N-recognin) of the Saccharomyces cerevisiae proteolytic system termed the proline (Pro)/N-degron pathway. Gid4 is a subunit of the oligomeric glucose-induced degradation (GID) ubiquitin ligase. Gid4 targets proteins through the binding to their Nt-Pro residue. Gid4 is also required for degradation of Nt-Xaa-Pro (Xaa is any amino acid residue) proteins such as Nt-[Ala-Pro]-Aro10 and Nt-[Ser-Pro]-Pck1, with Pro at position 2. Here, we show that specific aminopeptidases function as components of the Pro/N-degron pathway by removing Nt-Ala or Nt-Ser and yielding Nt-Pro, which can be recognized by Gid4-GID. Nt-Ala is removed by the previously uncharacterized aminopeptidase Fra1. The enzymatic activity of Fra1 is shown to be essential for the GID-dependent degradation of Nt-[Ala-Pro]-Aro10. Fra1 can also trim Nt-[Ala-Pro-Pro-Pro] (stopping immediately before the last Pro) and thereby can target for degradation a protein bearing this Nt sequence. Nt-Ser is removed largely by the mitochondrial/cytosolic/nuclear aminopeptidase Icp55. These advances are relevant to eukaryotes from fungi to animals and plants, as Fra1, Icp55, and the GID ubiquitin ligase are conserved in evolution. In addition to discovering the mechanism of targeting of Xaa-Pro proteins, these insights have also expanded the diversity of substrates of the Pro/N-degron pathway.

Keywords: Fra1; Icp55; aminopeptidase; degron; ubiquitin

DOI: https://doi.org/10.1073/pnas.2115430118

Autophagy. 2021 Oct 17. 1-3

The pro-autophagic protein AMBRA1 coordinates cell cycle progression by regulating CCND (cyclin D) stability.

Emiliano Maiani, Giacomo Milletti, Francesco Cecconi.

The scaffold protein AMBRA1 regulates the early steps of autophagosome formation and cell growth, and its deficiency is associated with neurodevelopmental defects and cancer. In a recent study, we show that AMBRA1 is a key factor in the upstream branch of the MYCN-MYC and CDK4-CDK6-dependent regulation of G1/S phase transition. Indeed, in the developing neuroepithelium, in neural stem cells, and in cancer cells, we demonstrate that AMBRA1 regulates the expression of D-type cyclins by controlling both their proteasomal degradation and their MYCN-MYC-mediated transcription. Also, we show that this regulation axis maintains genome integrity during DNA replication, and we identify a possible line of treatment for tumors downregulating AMBRA1 and/or overexpressing CCND1 (cyclin D1), by demonstrating that AMBRA1-depleted cells carry an AMBRA1-loss-specific lethal sensitivity to CHEK1 inhibition. Interestingly, we show that this aspect is specific for AMBRA1 loss, because ATG7 knockdown does not display the same response to CHEK1 inhibitors. Hence, our findings underscore that the AMBRA1-CCND1 pathway represents a novel crucial mechanism of cell cycle regulation, deeply interconnected with genomic stability in development and cancer.

Keywords: AMBRA1; cancer; cell cycle regulation; cyclin D1; neurodevelopment; replication stress; synthetic lethality

DOI: https://doi.org/10.1080/15548627.2021.1985917

Nat Commun. 2021 Oct 20. 12(1): 6101

GAK and PRKCD are positive regulators of PRKN-independent mitophagy.

Michael J Munson, Benan J Mathai, Matthew Yoke Wui Ng, Laura Trachsel-Moncho, Laura R de la Ballina, Sebastian W Schultz, Yahyah Aman, Alf H Lystad, Sakshi Singh, Sachin Singh, Jørgen Wesche, Evandro F Fang, Anne Simonsen.

The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive. Here, we have screened for regulators of PRKN-independent mitophagy using an siRNA library targeting 197 proteins containing lipid interacting domains. We identify Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCD) as regulators of PRKN-independent mitophagy, with both being dispensable for PRKN-dependent mitophagy and starvation-induced autophagy. We demonstrate that the kinase activity of both GAK and PRKCD are required for efficient mitophagy in vitro, that PRKCD is present on mitochondria, and that PRKCD facilitates recruitment of ULK1/ATG13 to early autophagic structures. Importantly, we demonstrate in vivo relevance for both kinases in the regulation of basal mitophagy. Knockdown of GAK homologue (gakh-1) in C. elegans or knockout of PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy regulation.

DOI: https://doi.org/10.1038/s41467-021-26331-7

Cell Death Dis. 2021 Oct 18. 12(11): 958

Relationship between lysosomal dyshomeostasis and progression of diabetic kidney disease.

Man Wu, Minjie Zhang, Yaozhi Zhang, Zixian Li, Xingyu Li, Zejian Liu, Huafeng Liu, Xiaoyu Li.

Lysosomes are organelles involved in cell metabolism, waste degradation, and cellular material circulation. They play a key role in the maintenance of cellular physiological homeostasis. Compared with the lysosomal content of other organs, that of the kidney is abundant, and lysosomal abnormalities are associated with the occurrence and development of certain renal diseases. Lysosomal structure and function in intrinsic renal cells are impaired in diabetic kidney disease (DKD). Promoting lysosomal biosynthesis and/or restoring lysosomal function can repair damaged podocytes and proximal tubular epithelial cells, and delay the progression of DKD. Lysosomal homeostasis maintenance may be advantageous in alleviating DKD. Here, we systematically reviewed the latest advances in the relationship between lysosomal dyshomeostasis and progression of DKD based on recent literature to further elucidate the mechanism of renal injury in diabetes mellitus and to highlight the application potential of lysosomal homeostasis maintenance as a new prevention and treatment strategy for DKD. However, research on screening effective interventions for lysosomal dyshomeostasis is still in its infancy, and thus should be the focus of future research studies. The screening out of cell-specific lysosomal function regulation targets according to the different stages of DKD, so as to realize the controllable targeted regulation of cell lysosomal function during DKD, is the key to the successful clinical development of this therapeutic strategy.

DOI: https://doi.org/10.1038/s41419-021-04271-w

Autophagy. 2021 Oct 17. 1-20

m6A reader YTHDC1 modulates autophagy by targeting SQSTM1 in diabetic skin.

Diefei Liang, Wei-Jye Lin, Meng Ren, Junxiong Qiu, Chuan Yang, Xiaoyi Wang, Na Li, Tingting Zeng, Kan Sun, Lili You, Li Yan, Wei Wang.

Dysregulation of macroautophagy/autophagy contributes to the delay of wound healing in diabetic skin. N6-methyladenosine (m6A) RNA modification is known to play a critical role in regulating autophagy. In this study, it was found that SQSTM1/p62 (sequestosome 1), an autophagy receptor, was significantly downregulated in two human keratinocyte cells lines with short-term high-glucose treatment, as well as in the epidermis of diabetic patients and a db/db mouse model with long-term hyperglycemia. Knockdown of SQSTM1 led to the impairment of autophagic flux, which was consistent with the results of high-glucose treatment in keratinocytes. Moreover, the m6A reader protein YTHDC1 (YTH domain containing 1), which interacted with SQSTM1 mRNA, was downregulated in keratinocytes under both the acute and chronic effects of hyperglycemia. Knockdown of YTHDC1 affected biological functions of keratinocytes, which included increased apoptosis rates and impaired wound-healing capacity. In addition, knockdown of endogenous YTHDC1 resulted in a blockade of autophagic flux in keratinocytes, while overexpression of YTHDC1 rescued the blockade of autophagic flux induced by high glucose. In vivo, knockdown of endogenous Ythdc1 or Sqstm1 inhibited autophagy in the epidermis and delayed wound healing. Interestingly, we found that a decrease of YTHDC1 drove SQSTM1 mRNA degradation in the nucleus. Furthermore, the results revealed that YTHDC1 interacted and cooperated with ELAVL1/HuR (ELAV like RNA binding protein 1) in modulating the expression of SQSTM1. Collectively, this study uncovered a previously unrecognized function for YTHDC1 in modulating autophagy via regulating the stability of SQSTM1 nuclear mRNA in diabetic keratinocytes.Abbreviations: ACTB: actin beta; AGEs: glycation end products; AL: autolysosome; AP: autophagosome; ATG: autophagy related; AKT: AKT serine/threonine kinase; ANOVA: analysis of variance; BECN1: beclin 1; Co-IP: co-immunoprecipitation; DEGs: differentially expressed genes; DM: diabetes mellitus; ELAVL1: ELAV like RNA binding protein 1; FTO: FTO alpha-ketoglutarate dependent dioxygenase; G: glucose; HaCaT: human keratinocyte; GO: Gene Ontology; GSEA: Gene Set Enrichment Analysis; HE: hematoxylin-eosin; IHC: immunohistochemical; IRS: immunoreactive score; KEAP1: kelch like ECH associated protein 1; KEGG: Kyoto Encyclopedia of Genes and Genomes; m6A: N6-methyladenosine; M: mannitol; MANOVA: multivariate analysis of variance; MAP1LC3: microtubule associated protein 1 light chain 3; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MeRIP: methylated RNA immunoprecipitation; METTL3: methyltransferase 3, N6-adenosine-methytransferase complex catalytic subunit; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; NG: normal glucose; NHEK: normal human epithelial keratinocyte; OE: overexpressing; p-: phospho-; PI: propidium iodide; PPIN: Protein-Protein Interaction Network; RBPs: RNA binding proteins; RIP: RNA immunoprecipitation; RNA-seq: RNA-sequence; RNU6-1: RNA, U6 small nuclear 1; ROS: reactive oxygen species; siRNAs: small interfering RNAs; SQSTM1: sequestosome 1; SRSF: serine and arginine rich splicing factor; T2DM: type 2 diabetes mellitus; TEM: transmission electron microscopy; TUBB: tubulin beta class I; WT: wild-type; YTHDC1: YTH domain containing 1.

Keywords: Autophagy; N6-methyladenosine; RNA stability; YTHDC1; diabetes; wound healing

DOI: https://doi.org/10.1080/15548627.2021.1974175

Nat Commun. 2021 Oct 22. 12(1): 6144

RIPK1 regulates starvation resistance by modulating aspartate catabolism.

Xinyu Mei, Yuan Guo, Zhangdan Xie, Yedan Zhong, Xiaofen Wu, Daichao Xu, Ying Li, Nan Liu, Zheng-Jiang Zhu.

RIPK1 is a crucial regulator of cell death and survival. Ripk1 deficiency promotes mouse survival in the prenatal period while inhibits survival in the early postnatal period without a clear mechanism. Metabolism regulation and autophagy are critical to neonatal survival from severe starvation at birth. However, the mechanism by which RIPK1 regulates starvation resistance and survival remains unclear. Here, we address this question by discovering the metabolic regulatory role of RIPK1. First, metabolomics analysis reveals that Ripk1 deficiency specifically increases aspartate levels in both mouse neonates and mammalian cells under starvation conditions. Increased aspartate in Ripk1-/- cells enhances the TCA flux and ATP production. The energy imbalance causes defective autophagy induction by inhibiting the AMPK/ULK1 pathway. Transcriptional analyses demonstrate that Ripk1-/- deficiency downregulates gene expression in aspartate catabolism by inactivating SP1. To summarize, this study reveals that RIPK1 serves as a metabolic regulator responsible for starvation resistance.

DOI: https://doi.org/10.1038/s41467-021-26423-4