bims-apauto 2022-11-13 papers

bims-apauto

Biomed News

on Apoptosis and autophagy

Issue of 2022–11–13
seven papers selected by
Su Hyun Lee, Seoul National University

Real-Time Deubiquitination Assays Using a Free Ubiquitin Sensor.
How cells recognize and remove the perforated lysosome.
Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy.
Selective PROTAC-mediated degradation of SMARCA2 is efficacious in SMARCA4 mutant cancers.
ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation.
Modulating autophagy and mitophagy as a promising therapeutic approach in neurodegenerative disorders.
Design and Synthesis of Ubiquitin-Based Chemical Tools with Unnatural Amino Acids for Selective Detection of Deubiquitinases.

Methods Mol Biol. 2023 ;2591 255-267

Real-Time Deubiquitination Assays Using a Free Ubiquitin Sensor.

Yun-Seok Choi, Robert E Cohen.

  Deubiquitinating enzymes cleave ubiquitin (Ub) from its attachment to another Ub, other proteins, peptides, or non-peptide adducts. In all cases, substrate hydrolysis by DUBs releases free Ub or polyubiquitin (polyUb) chains. Whereas most quantitative DUB assays depend on fluorescently labeled artificial substrates, employing a sensor able to detect Ub release in real time makes it possible to monitor DUB activity using virtually any Ub conjugate as a substrate. The protocols here describe the preparation of Atto532-tUI, a high-affinity sensor for free Ub, and its use in real-time deubiquitination assays.

Keywords:  DUB assay; Deubiquitinase; Deubiquitination; Fluorescent ubiquitin sensor; Ubiquitin; tUI

DOI:  https://doi.org/10.1007/978-1-0716-2803-4_15
Autophagy. 2022 Nov 11. 1-3

How cells recognize and remove the perforated lysosome.

Keisuke Tabata, Marika Saeki, Tamotsu Yoshimori, Maho Hamasaki.

  Macroautophagy (hereafter autophagy) is a highly conserved intracellular degradation system to maintain cellular homeostasis by degrading cellular components such as misfolded proteins, nonfunctional organelles, pathogens, and cytosol. Conversely, selective autophagy targets and degrades specific cargo, such as organelles, bacteria, etc. We previously reported that damaged lysosomes are autophagy targets, via a process called lysophagy. However, how cells target damaged lysosomes through autophagy is not known. We performed proteomics analysis followed by siRNA screening to identify genes involved in targeting damaged lysosomes and identified a new E3 ligase complex, involving CUL4A (cullin 4A), as a regulatory complex in lysophagy. We also found that this complex mediates K48-linked poly-ubiquitination on lysosome protein LAMP2 during lysosomal damage; particularly, the lumenal side of LAMP2 is important to recruit the complex to damaged lysosomes. This protein modification is thus critical to initiate the clearance of damaged lysosomes.

Keywords:  CUL4A; LAMP2; lysophagy; lysosomal membrane damage; selective autophagy; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2022.2138686
Cell Death Dis. 2022 Nov 10. 13(11): 947

Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy.

Nur Mehpare Kocaturk, Nesibe Peker, Karin Eberhart, Yunus Akkoc, Gamze Deveci, Joern Dengjel, Devrim Gozuacik.

Autophagy is an evolutionarily conserved eukaryotic cellular mechanism through which cytosolic fragments, misfolded/aggregated proteins and organelles are degraded and recycled. Priming of mitochondria through ubiquitylation is required for the clearance the organelle by autophagy (mitophagy). Familial Parkinson's Disease-related proteins, including the E3-ligase PARK2 (PARKIN) and the serine/threonine kinase PARK6 (PINK1) control these ubiquitylation reactions and contribute to the regulation of mitophagy. Here we describe, novel protein complexes containing autophagy protein ATG5 and ubiquitin-proteasome system (UPS) components. We discovered that ATG5 interacts with PSMA7 and PARK2 upon mitochondrial stress. Results suggest that all three proteins translocate mitochondria and involve in protein complexes containing autophagy, UPS and mitophagy proteins. Interestingly, PARK2 and ATG5 recruitment onto mitochondria requires proteasome components PSMA7 and PSMB5. Strikingly, we discovered that subunit of 20 S proteasome, PSMA7, is required for the progression of PARK2-PARK6-mediated mitophagy and the proteasome activity following mitochondrial stress. Our results demonstrate direct, dynamic and functional interactions between autophagy and UPS components that contribute to the regulation of mitophagy.

DOI: https://doi.org/10.1038/s41419-022-05339-x
Nat Commun. 2022 Nov 10. 13(1): 6814

Selective PROTAC-mediated degradation of SMARCA2 is efficacious in SMARCA4 mutant cancers.

Jennifer Cantley, Xiaofen Ye, Emma Rousseau, Tom Januario, Brian D Hamman, Christopher M Rose, Tommy K Cheung, Trent Hinkle, Leofal Soto, Connor Quinn, Alicia Harbin, Elizabeth Bortolon, Xin Chen, Roy Haskell, Eva Lin, Shang-Fan Yu, Geoff Del Rosario, Emily Chan, Debra Dunlap, Hartmut Koeppen, Scott Martin, Mark Merchant, Matt Grimmer, Fabio Broccatelli, Jing Wang, Jennifer Pizzano, Peter S Dragovich, Michael Berlin, Robert L Yauch.

The mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) helicase SMARCA4 is frequently mutated in cancer and inactivation results in a cellular dependence on its paralog, SMARCA2, thus making SMARCA2 an attractive synthetic lethal target. However, published data indicates that achieving a high degree of selective SMARCA2 inhibition is likely essential to afford an acceptable therapeutic index, and realizing this objective is challenging due to the homology with the SMARCA4 paralog. Herein we report the discovery of a potent and selective SMARCA2 proteolysis-targeting chimera molecule (PROTAC), A947. Selective SMARCA2 degradation is achieved in the absence of selective SMARCA2/4 PROTAC binding and translates to potent in vitro growth inhibition and in vivo efficacy in SMARCA4 mutant models, compared to wild type models. Global ubiquitin mapping and proteome profiling reveal no unexpected off-target degradation related to A947 treatment. Our study thus highlights the ability to transform a non-selective SMARCA2/4-binding ligand into a selective and efficacious in vivo SMARCA2-targeting PROTAC, and thereby provides a potential new therapeutic opportunity for patients whose tumors contain SMARCA4 mutations.

DOI: https://doi.org/10.1038/s41467-022-34562-5
Mol Cell. 2022 Nov 01. pii: S1097-2765(22)01017-6. [Epub ahead of print]

ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation.

Alexander R van Vliet, George N Chiduza, Sarah L Maslen, Valerie E Pye, Dhira Joshi, Stefano De Tito, Harold B J Jefferies, Evangelos Christodoulou, Chloë Roustan, Emma Punch, Javier H Hervás, Nicola O'Reilly, J Mark Skehel, Peter Cherepanov, Sharon A Tooze.

  ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.

Keywords:  AlphaFold; autophagosome; autophagy; integrative structure prediction; lipid scramblase; lipid transfer

DOI:  https://doi.org/10.1016/j.molcel.2022.10.017
Life Sci. 2022 Nov 04. pii: S0024-3205(22)00853-0. [Epub ahead of print] 121153

Modulating autophagy and mitophagy as a promising therapeutic approach in neurodegenerative disorders.

Jayapriya Mishra, Gurjit Kaur Bhatti, Abhishek Sehrawat, Charan Singh, Arti Singh, Arubala P Reddy, P Hemachandra Reddy, Jasvinder Singh Bhatti.

  The high prevalence of neurodegenerative diseases has become a major public health challenge and is associated with a tremendous burden on individuals, society and federal governments worldwide. Protein misfolding and aggregation are the major pathological hallmarks of several neurodegenerative disorders. The cells have evolved several regulatory mechanisms to deal with aberrant protein folding, namely the classical ubiquitin pathway, where ubiquitination of protein aggregates marks their degradation via lysosome and the novel autophagy or mitophagy pathways. Autophagy is a catabolic process in eukaryotic cells that allows the lysosome to recycle the cell's own contents, such as organelles and proteins, known as autophagic cargo. Their most significant role is to keep cells alive in distressed situations. Mitophagy is also crucial for reducing abnormal protein aggregation and increasing organelle clearance and partly accounts for maintaining cellular homeostasis. Furthermore, substantial data indicate that any disruption in these homeostatic mechanisms leads to the emergence of several age-associated metabolic and neurodegenerative diseases. So, targeting autophagy and mitophagy might be a potential therapeutic strategy for a variety of health conditions.

Keywords:  Aggregation; Autophagic cargo; Autophagy; Functional foods; Homeostasis; Misfolding; Mitophagy; Neurodegenerative disorders

DOI:  https://doi.org/10.1016/j.lfs.2022.121153
Methods Mol Biol. 2023 ;2591 59-78

Design and Synthesis of Ubiquitin-Based Chemical Tools with Unnatural Amino Acids for Selective Detection of Deubiquitinases.

Wioletta Rut, Mikolaj Zmudzinski, Marcin Drag.

  Several chemical approaches have been applied to develop Ub-based substrates and probes selective toward one or a narrow subset of deubiquitinases (DUBs). Since DUBs are highly specific toward ubiquitin and exhibit low activity toward shorter peptides, it is challenging to design truly selective chemical tools to investigate one DUB in biological samples. Incorporating amino acids other than canonical LRG at the P4-P2 positions in the Ub improves DUB activity and selectivity toward Ub derivatives. Here, we describe the protocol for identifying selective peptide sequences using a hybrid combinatorial substrate library (HyCoSuL) approach that can be introduced in the C-terminal motif of Ub. Furthermore, we describe the synthesis protocol of Ub-based probes and substrates containing unnatural amino acids and the application of Ub-based probes to detect DUBs in cell lysates.

Keywords:  Combinatorial library; DUBs; Substrate specificity; Ubiquitin; Unnatural amino acids

DOI:  https://doi.org/10.1007/978-1-0716-2803-4_5