bims-apauto 2023-04-09 papers

Issue of 2023–04–09
six papers selected by
Su Hyun Lee, Harvard University

Methods Mol Biol. 2023 ;2620 243-252

Monitoring the Activation of Selective Autophagy via N-Terminal Arginylation.

Chang Hoon Ji, Su Bin Kim, Min Ju Lee, Yong Tae Kwon.

In addition to generating N-degron-carrying substrates destined for proteolysis, N-terminal arginylation can globally upregulate selective macroautophagy via activation of the autophagic N-recognin and archetypal autophagy cargo receptor p62/SQSTM1/sequestosome-1. To evaluate the macroautophagic turnover of cellular substrates, including protein aggregates (aggrephagy) and subcellular organelles (organellophagy) mediated by N-terminal arginylation in vivo, we report here a protocol for assaying the activation of the autophagic Arg/N-degron pathway and degradation of cellular cargoes via N-terminal arginylation. These methods, reagents, and conditions are applicable across a wide spectrum of different cell lines, primary cultures, and/or animal tissues, thereby providing a general means for identification and validation of putative cellular cargoes degraded by Nt-arginylation-activated selective autophagy.

Keywords: ATE1; Autophagic flux; Detergent-insoluble/soluble fractionation; In vitro p62/SQSTM1 self-oligomerization; Nt-arginylation; Punctate formation/co-localization

DOI: https://doi.org/10.1007/978-1-0716-2942-0_26

Autophagy. 2023 Apr 04.

LC3-independent autophagy is vital to prevent TNF cytotoxicity.

Dario Priem, Jon Huyghe, Mathieu Jm Bertrand.

The (macro)autophagy field is facing a paradigm shift after the recent discovery that cytosolic cargoes can still be selectively targeted to phagophores (the precursors to autophagosomes) even in the absence of LC3 or other Atg8-protein family members. Several in vitro studies have indeed reported on the existence of an unconventional selective autophagic pathway that involves the in-situ formation of an autophagosome around the cargo through the direct selective autophagy receptor-mediated recruitment of RB1CC1/FIP200, thereby bypassing the requirement of LC3. In an article recently published in Science, we demonstrate the physiological importance of this unconventional autophagic pathway in the context of TNF (tumor necrosis factor) signaling. We show that it promotes the degradation of the cytotoxic TNFRSF1A/TNFR1 (TNF receptor superfamily member 1A) complex II that assembles upon TNF sensing and thereby protects mice from TNFRSF1A-driven embryonic lethality and skin inflammation.

Keywords: ATG9A; Apoptosis; LC3-independent autophagy; RB1CC1; TAX1BP1; cell death; embryogenesis; mouse development; skin disease; unconventional autophagy

DOI: https://doi.org/10.1080/15548627.2023.2197760

Cancer Sci. 2023 Apr 03.

Autophagy and cancer: basic mechanisms and inhibitor development.

Yutaro Hama, Yuta Ogasawara, Nobuo N Noda.

Autophagy is a lysosomal degradation system of cytoplasmic components, and it contributes to cellular homeostasis through turnover of various biomolecules and organelles, often in a selective manner. Autophagy is closely related to cancer, but its roles in cancer are complicated. It works as either a promoter or suppressor, depending on the stage and type of cancer. In this review, we briefly summarize the basic mechanisms of autophagy and describe the complicated roles of autophagy in cancer. Moreover, we summarize the clinical trials of autophagy inhibitors targeting cancer and the development of more specific autophagy inhibitors for future clinical application.

Keywords: ATG; autophagy; cancer; chloroquine; inhibitor; mTORC1

DOI: https://doi.org/10.1111/cas.15803

Nat Commun. 2023 Apr 05. 14(1): 1906

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing.

Zhen-Dong Zhong, Ying-Yuan Xie, Hong-Xuan Chen, Ye-Lin Lan, Xue-Hong Liu, Jing-Yun Ji, Fu Wu, Lingmei Jin, Jiekai Chen, Daniel W Mak, Zhang Zhang, Guan-Zheng Luo.

N6-methyladenosine (m6A) has been increasingly recognized as a new and important regulator of gene expression. To date, transcriptome-wide m6A detection primarily relies on well-established methods using next-generation sequencing (NGS) platform. However, direct RNA sequencing (DRS) using the Oxford Nanopore Technologies (ONT) platform has recently emerged as a promising alternative method to study m6A. While multiple computational tools are being developed to facilitate the direct detection of nucleotide modifications, little is known about the capabilities and limitations of these tools. Here, we systematically compare ten tools used for mapping m6A from ONT DRS data. We find that most tools present a trade-off between precision and recall, and integrating results from multiple tools greatly improve performance. Using a negative control could improve precision by subtracting certain intrinsic bias. We also observed variation in detection capabilities and quantitative information among motifs, and identified sequencing depth and m6A stoichiometry as potential factors affecting performance. Our study provides insight into the computational tools currently used for mapping m6A based on ONT DRS data and highlights the potential for further improving these tools, which may serve as the basis for future research.

DOI: https://doi.org/10.1038/s41467-023-37596-5

Apoptosis. 2023 Apr 04.

Apoptotic proteins with non-apoptotic activity: expression and function in cancer.

Varda Shoshan-Barmatz, Tasleem Arif, Anna Shteinfer-Kuzmine.

Apoptosis is a process of programmed cell death in which a cell commits suicide while maintaining the integrity and architecture of the tissue as a whole. Apoptosis involves activation of one of two major pathways: the extrinsic pathway, where extracellular pro-apoptotic signals, transduced through plasma membrane death receptors, activate a caspase cascade leading to apoptosis. The second, the intrinsic apoptotic pathway, where damaged DNA, oxidative stress, or chemicals, induce the release of pro-apoptotic proteins from the mitochondria, leading to the activation of caspase-dependent and independent apoptosis. However, it has recently become apparent that proteins involved in apoptosis also exhibit non-cell death-related physiological functions that are related to the cell cycle, differentiation, metabolism, inflammation or immunity. Such non-conventional activities were predominantly reported in non-cancer cells although, recently, such a dual function for pro-apoptotic proteins has also been reported in cancers where they are overexpressed. Interestingly, some apoptotic proteins translocate to the nucleus in order to perform a non-apoptotic function. In this review, we summarize the unconventional roles of the apoptotic proteins from a functional perspective, while focusing on two mitochondrial proteins: VDAC1 and SMAC/Diablo. Despite having pro-apoptotic functions, these proteins are overexpressed in cancers and this apparent paradox and the associated pathophysiological implications will be discussed. We will also present possible mechanisms underlying the switch from apoptotic to non-apoptotic activities although a deeper investigation into the process awaits further study.

Keywords: Apoptosis; Cancer; Metabolism; Mitochondria; SMAC/Diablo; VDAC1

DOI: https://doi.org/10.1007/s10495-023-01835-3

Cell. 2023 Mar 31. pii: S0092-8674(23)00213-1. [Epub ahead of print]

Systemwide disassembly and assembly of SCF ubiquitin ligase complexes.

Kheewoong Baek, Daniel C Scott, Lukas T Henneberg, Moeko T King, Matthias Mann, Brenda A Schulman.

Cells respond to environmental cues by remodeling their inventories of multiprotein complexes. Cellular repertoires of SCF (SKP1-CUL1-F box protein) ubiquitin ligase complexes, which mediate much protein degradation, require CAND1 to distribute the limiting CUL1 subunit across the family of ∼70 different F box proteins. Yet, how a single factor coordinately assembles numerous distinct multiprotein complexes remains unknown. We obtained cryo-EM structures of CAND1-bound SCF complexes in multiple states and correlated mutational effects on structures, biochemistry, and cellular assays. The data suggest that CAND1 clasps idling catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF. New SCF production proceeds in reverse, through SKP1-F box allosterically destabilizing CAND1. The CAND1-SCF conformational ensemble recycles CUL1 from inactive complexes, fueling mixing and matching of SCF parts for E3 activation in response to substrate availability. Our data reveal biogenesis of a predominant family of E3 ligases, and the molecular basis for systemwide multiprotein complex assembly.

Keywords: CAND1; CRL; E3 ligase; NEDD8; SCF; cryo-EM; cullin-RING ligase; protein complex assembly; proteomics; ubiquitin

DOI: https://doi.org/10.1016/j.cell.2023.02.035