bims-apauto 2021-12-05 papers

bims-apauto

Biomed News

on Apoptosis and autophagy

Issue of 2021–12–05
eleven papers selected by
Su Hyun Lee, Seoul National University

USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1.
The caspase-6-p62 axis modulates p62 droplets based autophagy in a dominant-negative manner.
Isoforms of autophagy-related proteins: role in glioma progression and therapy resistance.
The complex interplay between ULK1 and protein phosphatases in autophagy regulation.
The unique Akt inhibitor SC66 suppressed AMPK activity and abolished autophagy through the EGFR-p62 pathway.
Vinexin contributes to autophagic decline in brain ageing across species.
Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis.
Arsenite-induced cytotoxicity is regulated by p38-SQSTM1/p62 and JNK-BNIP3L/Nix signaling in lung cancer cells.
USP10 inhibits the dopamine-induced reactive oxygen species-dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity.
Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1.
Ferroptosis mediates selective motor neuron death in amyotrophic lateral sclerosis.

Cell Death Differ. 2021 Nov 27.

USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1.

Yuluo Rong, Jin Fan, Chengyue Ji, Zhuanghui Wang, Xuhui Ge, Jiaxing Wang, Wu Ye, Guoyong Yin, Weihua Cai, Wei Liu.

Spinal cord ischemia-reperfusion injury (SCIRI) is a serious trauma that can lead to loss of sensory and motor function. Ferroptosis is a new form of regulatory cell death characterized by iron-dependent accumulation of lipid peroxides. Ferroptosis has been studied in various diseases; however, the exact function and molecular mechanism of ferroptosis in SCIRI remain unknown. In this study, we demonstrated that ferroptosis is involved in the pathological mechanism of SCIRI. Inhibition of ferroptosis could promote the recovery of motor function in mice after SCIRI. In addition, we found that ubiquitin-specific protease 11 (USP11) was significantly upregulated in neuronal cells after hypoxia-reoxygenation and in the spinal cord in mice with I/R injury. Knockdown of USP11 in vitro and KO of USP11 in vivo (USP11-/Y) significantly decreased neuronal cell ferroptosis. In mice, this promotes functional recovery after SCIRI. In contrast, in vitro, USP11 overexpression leads to classic ferroptosis events. Overexpression of USP11 in mice resulted in increased ferroptosis and poor functional recovery after SCIRI. Interestingly, upregulating the expression of USP11 also appeared to increase the production of autophagosomes and to cause substantial autophagic flux, a potential mechanism through which USP11 may enhance ferroptosis. The decreased autophagy markedly weakened the ferroptosis mediated by USP11 and autophagy induction had a synergistic effect with USP11. Importantly, USP11 promotes autophagy activation by stabilizing Beclin 1, thereby leading to ferroptosis. In conclusion, this study shows that ferroptosis is closely associated with SCIRI, and that USP11 plays a key role in regulating ferroptosis and additionally identifies USP11-mediated autophagy-dependent ferroptosis as a promising target for the treatment of SCIRI.

DOI: https://doi.org/10.1038/s41418-021-00907-8
Cell Death Differ. 2021 Dec 03.

The caspase-6-p62 axis modulates p62 droplets based autophagy in a dominant-negative manner.

Evelina Valionyte, Yi Yang, Sophie A Griffiths, Amelia T Bone, Elizabeth R Barrow, Vikram Sharma, Boxun Lu, Shouqing Luo.

SQSTM1/p62, as a major autophagy receptor, forms droplets that are critical for cargo recognition, nucleation, and clearance. p62 droplets also function as liquid assembly platforms to allow the formation of autophagosomes at their surfaces. It is unknown how p62-droplet formation is regulated under physiological or pathological conditions. Here, we report that p62-droplet formation is selectively blocked by inflammatory toxicity, which induces cleavage of p62 by caspase-6 at a novel cleavage site D256, a conserved site across human, mouse, rat, and zebrafish. The N-terminal cleavage product is relatively stable, whereas the C-terminal product appears undetectable. Using a variety of cellular models, we show that the p62 N-terminal caspase-6 cleavage product (p62-N) plays a dominant-negative role to block p62-droplet formation. In vitro p62 phase separation assays confirm this observation. Dominant-negative regulation of p62-droplet formation by caspase-6 cleavage attenuates p62 droplets dependent autophagosome formation. Our study suggests a novel pathway to modulate autophagy through the caspase-6-p62 axis under certain stress stimuli.

DOI: https://doi.org/10.1038/s41418-021-00912-x
Mol Cell Biochem. 2021 Dec 01.

Isoforms of autophagy-related proteins: role in glioma progression and therapy resistance.

Elizaveta Belyaeva, Rajesh Kumar Kharwar, Ilya V Ulasov, Irina Karlina, Petr Timashev, Reza Mohammadinejad, Arbind Acharya.

  Autophagy is the process of recycling and utilization of degraded organelles and macromolecules in the cell compartments formed during the fusion of autophagosomes with lysosomes. During autophagy induction the healthy and tumor cells adapt themselves to harsh conditions such as cellular stress or insufficient supply of nutrients in the cell environment to maintain their homeostasis. Autophagy is currently seen as a form of programmed cell death along with apoptosis and necroptosis. In recent years multiple studies have considered the autophagy as a potential mechanism of anticancer therapy in malignant glioma. Although, subsequent steps in autophagy development are known and well-described, on molecular level the mechanism of autophagosome initiation and maturation using autophagy-related proteins is under investigation. This article reviews current state about the mechanism of autophagy, its molecular pathways and the most recent studies on roles of autophagy-related proteins and their isoforms in glioma progression and its treatment.

Keywords:  Autophagy; Glioblastoma; Isoforms; Tumor resistance

DOI:  https://doi.org/10.1007/s11010-021-04308-w
Autophagy. 2021 Nov 28. 1-2

The complex interplay between ULK1 and protein phosphatases in autophagy regulation.

Devanarayanan Siva Sankar, Zehan Hu, Joern Dengjel.

  ULK1 kinase is the gatekeeper of canonical macroautophagy (hereafter referred to as autophagy) phosphorylating an array of substrates critical for autophagosome biogenesis. To uncover if ULK1 has broader functions also regulating subsequent steps of autophagosome turnover, i.e., maturation, lysosomal fusion, and degradation, we performed a set of unbiased phosphoproteomic experiments employing mouse and human cells in combination with genetic and environmental perturbations. We characterized more than 1,000 potential ULK1 target sites of which many affect proteins known to be involved in all phases of the autophagosome life cycle. To better understand which of these 1,000 phosphosites were directly phosphorylated by ULK1, in contrast to downstream kinases being activated or phosphatases being inhibited by ULK1, we developed a proteome-scale in vitro kinase assay and characterized 187 phosphosites on 157 proteins as bona fide ULK1 target sites. Interestingly, our results highlight an intricate crosstalk between ULK1 and protein phosphatases. Focusing on STRN (striatin), a regulatory subunit of PPP2/PP2A (protein phosphatase 2), we identified a positive feedback loop linked to ULK1 and promoting autophagy.

Keywords:  Feedback; STRIPAK; STRN; ULK1; in vitro kinase assay; kinase; phosphatase; phosphoproteomics; phosphorylation; striatin

DOI:  https://doi.org/10.1080/15548627.2021.2002546
Cell Biol Int. 2021 Dec 02.

The unique Akt inhibitor SC66 suppressed AMPK activity and abolished autophagy through the EGFR-p62 pathway.

Bolin Hou, Erwei Li, Jingnan Liang, Shuchun Liu, Huaiyi Yang, Ling Liu, Xuejun Jiang.

  Akt is usually considered to be a negative regulator of both autophagy and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling. In the present study, we found that SC66, a pyridine-based allosteric Akt inhibitor, suppressed basal and H2 O2 -induced autophagy concurrent with decreased phosphorylation and activity of AMPK. SC66 treatment led to the formation of a high molecular weight (HMW) form of SQSTM1/p62 (p62), which is an autophagic substrate and is essential for selective autophagy. Moreover, we observed that SC66 inhibited the binding of p62 and microtubule-associated protein light chain 3 (LC3). The immunoprecipitation results revealed the interaction between p62 and epidermal growth factor receptor (EGFR), and knockdown of EGFR reversed SC66-mediated autophagy inhibition without affecting the phosphorylation of acetyl-CoA carboxylase (ACC), a well-known substrate of AMPK. SC66 increased the interaction between EGFR and Beclin 1 and markedly decreased the association of EGFR with VPS34, a critical protein for autophagy induction. Collectively, the data presented here indicate that EGFR-p62 pathway plays a critical role in Akt-mediated positive regulation of autophagy. This article is protected by copyright. All rights reserved.

Keywords:  AMPK; EGFR; SC66; autophagy; p62

DOI:  https://doi.org/10.1002/cbin.11732
Cell Death Differ. 2021 Nov 30.

Vinexin contributes to autophagic decline in brain ageing across species.

So Jung Park, Rebecca A Frake, Cansu Karabiyik, Sung Min Son, Farah H Siddiqi, Carla F Bento, Peter Sterk, Mariella Vicinanza, Mariana Pavel, David C Rubinsztein.

Autophagic decline is considered a hallmark of ageing. The activity of this intracytoplasmic degradation pathway decreases with age in many tissues and autophagy induction ameliorates ageing in many organisms, including mice. Autophagy is a critical protective pathway in neurons and ageing is the primary risk factor for common neurodegenerative diseases. Here, we describe that autophagosome biogenesis declines with age in mouse brains and that this correlates with increased expression of the SORBS3 gene (encoding vinexin) in older mouse and human brain tissue. We characterise vinexin as a negative regulator of autophagy. SORBS3 knockdown increases F-actin structures, which compete with YAP/TAZ for binding to their negative regulators, angiomotins, in the cytosol. This promotes YAP/TAZ translocation into the nucleus, thereby increasing YAP/TAZ transcriptional activity and autophagy. Our data therefore suggest brain autophagy decreases with age in mammals and that this is likely, in part, mediated by increasing levels of vinexin.

DOI: https://doi.org/10.1038/s41418-021-00903-y
Nat Commun. 2021 Nov 30. 12(1): 6984

Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis.

Maxime Uriarte, Nadine Sen Nkwe, Roch Tremblay, Oumaima Ahmed, Clémence Messmer, Nazar Mashtalir, Haithem Barbour, Louis Masclef, Marion Voide, Claire Viallard, Salima Daou, Djaileb Abdelhadi, Daryl Ronato, Mohammadjavad Paydar, Anaïs Darracq, Karine Boulay, Nicolas Desjardins-Lecavalier, Przemyslaw Sapieha, Jean-Yves Masson, Mikhail Sergeev, Benjamin H Kwok, Laura Hulea, Frédérick A Mallette, Eric Milot, Bruno Larrivée, Hugo Wurtele, El Bachir Affar.

Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions.

DOI: https://doi.org/10.1038/s41467-021-27306-4
Biochem Biophys Res Commun. 2021 Nov 26. pii: S0006-291X(21)01585-0. [Epub ahead of print]587 16-23

Arsenite-induced cytotoxicity is regulated by p38-SQSTM1/p62 and JNK-BNIP3L/Nix signaling in lung cancer cells.

Hyun-Young Lee, Seon-Hee Oh.

  Arsenic is a potent carcinogen in humans. However, the molecular mechanisms underlying its toxicity in lung cancer remain unclear. Here, we report that arsenite-induced cytotoxicity is regulated by SQSTM1/p62 and BNIP3L/Nix signaling in non-small-cell lung cancer H460 cells. Arsenite exposure resulted in dose-dependent growth inhibition, which was associated with apoptosis, as demonstrated by depolarized mitochondrial membrane potential and cleavage of caspase-8, caspase-3, PARP-1, and Bax. The autophagy adaptor p62 was detected in the monomeric and multiple high-molecular-weight (HMW) forms, and protein levels were upregulated depending on both arsenite concentrations (≤45 μM) and exposure times (<24 h). LC3-II, an autophagy marker, was upregulated as early as 1 h after arsenite treatment. Expression of Nix, a mitochondrial outer membrane protein, continued to increase with arsenite concentration and exposure time; it was detected in the monomeric and multiple HMW forms. Soon after arsenite exposure, p62 colocalized with Nix in the cytoplasm, and p62 knockdown reduced the Nix levels and increased the LC3-II levels. In contrast, Nix knockdown did not affect the p62 and LC3-II levels but reduced caspase-8, caspase-3, and Bax cleavage, indicating that Nix accumulation resulted from its reduced autophagic degradation and promoted apoptosis. p38 inhibition markedly increased arsenite-induced Nix protein and reduced p62 protein levels, resulting in increased autophagy and apoptosis. Furthermore, c-Jun NH2-terminal kinase inhibition reduced Nix and Bax cleavage, and both signaling pathways were suppressed by N-acetylcysteine treatment. Our results suggest that arsenite-induced cytotoxicity is modulated by the coordinated action of p62 and Nix through MAPK.

Keywords:  Arsenite; Autophagy; Bax cleavage; Nix; Non-small-cell lung cancer; p62

DOI:  https://doi.org/10.1016/j.bbrc.2021.11.068
J Biol Chem. 2021 Nov 24. pii: S0021-9258(21)01257-6. [Epub ahead of print] 101448

USP10 inhibits the dopamine-induced reactive oxygen species-dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity.

Junya Sango, Taichi Kakihana, Masahiko Takahashi, Yoshinori Katsuragi, Sergei Anisimov, Masaaki Komatsu, Masahiro Fujii.

  Nrf2 is an antioxidant transcriptional activator in many types of cells, and its dysfunction plays key roles in a variety of human disorders, including Parkinson's disease (PD). PD is characterized by the selective loss of dopaminergic neurons in PD-affected brain regions. Dopamine treatment of neuronal cells stimulates the production of reactive oxygen species (ROS) and increases ROS-dependent neuronal apoptosis. In this study, we found that the ubiquitin-specific protease 10 (USP10) protein reduces dopamine-induced ROS production of neuronal cells and ROS-dependent apoptosis by stimulating the antioxidant activity of Nrf2. USP10 interacted with the Nrf2 activator p62, increased the phosphorylation of p62, increased the interaction of p62 with the Nrf2 inhibitor Keap1, and stimulated Nrf2 antioxidant transcriptional activity. In addition, USP10 augmented dopamine-induced Nrf2 translation. Taken together, these results indicate that USP10 is a key regulator of Nrf2 antioxidant activity in neuronal cells and suggest that USP10 activators are promising therapeutic agents for oxidative stress-related diseases, including PD.

Keywords:  Keap1; Nrf2; Parkinson's disease; ROS; USP10; apoptosis; dopamine; p62

DOI:  https://doi.org/10.1016/j.jbc.2021.101448
J Extracell Vesicles. 2021 Dec;10(14): e12166

Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1.

Tom G Keulers, Sten F Libregts, Joel E J Beaumont, Kim G Savelkouls, Johan Bussink, Hans Duimel, Ludwig Dubois, Marijke I Zonneveld, Carmen López-Iglesias, Karel Bezstarosti, Jeroen A Demmers, Marc Vooijs, Marca Wauben, Kasper M A Rouschop.

  Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro-angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+ EVs with GABARAPL1 targeting antibodies results in blockade of pro-angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+ EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target.

Keywords:  GABARAPL1; autophagy; exosomes; extracellular vesicles; hypoxia

DOI:  https://doi.org/10.1002/jev2.12166
Cell Death Differ. 2021 Dec 02.

Ferroptosis mediates selective motor neuron death in amyotrophic lateral sclerosis.

Taide Wang, Doris Tomas, Nirma D Perera, Brittany Cuic, Sophia Luikinga, Aida Viden, Samantha K Barton, Catriona A McLean, André L Samson, Adam Southon, Ashley I Bush, James M Murphy, Bradley J Turner.

Amyotrophic lateral sclerosis (ALS) is caused by selective degeneration of motor neurons in the brain and spinal cord; however, the primary cell death pathway(s) mediating motor neuron demise remain elusive. We recently established that necroptosis, an inflammatory form of regulated cell death, was dispensable for motor neuron death in a mouse model of ALS, implicating other forms of cell death. Here, we confirm these findings in ALS patients, showing a lack of expression of key necroptotic effector proteins in spinal cords. Rather, we uncover evidence for ferroptosis, a recently discovered iron-dependent form of regulated cell death, in ALS. Depletion of glutathione peroxidase 4 (GPX4), an anti-oxidant enzyme and central repressor of ferroptosis, occurred in post-mortem spinal cords of both sporadic and familial ALS patients. GPX4 depletion was also an early and universal feature of spinal cords and brains of transgenic mutant superoxide dismutase 1 (SOD1G93A), TDP-43 and C9orf72 mouse models of ALS. GPX4 depletion and ferroptosis were linked to impaired NRF2 signalling and dysregulation of glutathione synthesis and iron-binding proteins. Novel BAC transgenic mice overexpressing human GPX4 exhibited high GPX4 expression localised to spinal motor neurons. Human GPX4 overexpression in SOD1G93A mice significantly delayed disease onset, improved locomotor function and prolonged lifespan, which was attributed to attenuated lipid peroxidation and motor neuron preservation. Our study discovers a new role for ferroptosis in mediating motor neuron death in ALS, supporting the use of anti-ferroptotic therapeutic strategies, such as GPX4 pathway induction and upregulation, for ALS treatment.

DOI: https://doi.org/10.1038/s41418-021-00910-z