Abstract 269: Duo-impairment of the Ubiquitin-Proteasome System and Autophagy by Ablation of COP9 Signalosome Subunit 8 Activates a Programmed Necrosis Pathway Mediated by RIP1-RIP3 Kinases but not Cyclophilin D-regulated Mitochondrial Membrane Permeability

Programmed cell death includes apoptosis and programmed necrosis (AKA, necroptosis). A well-recognized feature of necrosis is the loss of membrane integrity of the dying cell. Cells undergoing apoptosis, however, maintain their membrane integrity. We previously reported massive cardiomyocyte (CM) necrosis, before increased apoptosis is discernible, in mouse hearts with cardiomyocyte-restricted knockout of the COP9 signalosome subunit 8 (CR-Csn8KO). This is associated with defective autophagosome maturation and ubiquitin-proteasome system (UPS) dysfunction. Consequently, mice with perinatal CR-Csn8KO develop rapidly dilated cardiomyopathy (DCM) and die prematurely. The present study sought to search for the mechanisms underlying the CM necrosis. Here our immunoprecipitation revealed significant increases of RIP1-interacted RIP3 in CR-Csn8KO myocardium, indicative of activation of the RIP1-RIP3 pathway. The RIP1-RIP3 pathway is known to mediate necroptosis. To further test whether RIP1 activation plays an essential role in CM necrosis in CR-Csn8KO mice, we treated the mice with a RIP1 kinase specific inhibitor necrostatin-1 (Nec-1) or vehicle control via osmotic mini-pump implanted in the peritoneal cavity at 2 weeks of age. Nec-1 significantly suppressed CM necrosis as measured by the positivity of Evan’s blue dye (EBD) uptake (p<0.00001), prevented left ventricle dilatation (p<0.05) at 3 weeks, and delayed premature death of the CR-Csn8KO mice (p=0.0072). These results demonstrate that CM necrosis in CR-Csn8KO mice is necroptosis in which the RIP1-RIP3 kinases-mediated pathway plays a major pathogenic role, and that CM necroptosis is the primary cause of DCM and mouse premature death. Increased mitochondrial membrane permeability, which can be suppressed by inhibition of cyclophilin D, has been shown to play a critical mediating role in myocytes necrosis. Hence, we cross-bred CR-Csn8KO mice with cyclophilin D knockout mice and obtained the Kaplan-Meier survival curve. Unexpectedly, cyclophilin D deficiency exacerbated the premature death of CR-Csn8KO mice (p=0.007), indicating that cyclophilin D regulated mitochondrial membrane permeability does not play an important role in CM necrosis in CR-Csn8KO mouse hearts.

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Abstract P043: The COP9 Signalosome Regulates Autophagy

Circulation Research ◽

10.1161/res.109.suppl_1.ap043 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Huabo Su ◽

Faqian Li ◽

Mark J Ranek ◽

Ning Wei ◽

Xuejun (XJ) Wang

Keyword(s):

Premature Death ◽

Ubiquitin Proteasome System ◽

Reciprocal Relationship ◽

Cop9 Signalosome ◽

Autophagic Flux ◽

Selective Autophagy ◽

Protein Levels ◽

Evolutionarily Conserved ◽

Ubiquitin Proteasome ◽

Lc3 Puncta

By indiscriminately degrading portions of cytoplasm for self-supply of nutrients, non-selective autophagy helps the cell to survive starvation. Selective autophagy, however, removes defective/surplus organelles and protein aggregates, thereby playing an important role in quality control in the cell. A utophagy is involved in the pathophysiology of a variety of disease, including common forms of heart disease. Mechanisms regulating autophagy, especially selective autophagy, remain poorly understood. The COP9 signalosome (CSN) is an evolutionarily conserved protein complex consisting of 8 subunits (CSN1 through CSN8). CSN was purported to regulate ubiquitin-proteasome system (UPS) mediated proteolysis. We recently reported UPS malfunction and the accumulation of ubiquitin positive aggregates in the cardiomyocytes of mice with perinatal cardiomyocyte-restricted Csn8 knockout (CR-Csn8KO), which displayed massive cardiomyocyte necrosis, congestive heart failure, and premature death. Here we report that Csn8/CSN is required for the removal of autophagosomes in cardiomyocytes, an exciting discovery that has not been reported in any types of cells. CR-Csn8KO mouse hearts show marked increases in LC3-II, indicative of increased autophagosomes. The increase in LC-II is accompanied by a significant increase in p62 protein levels, which is evident as early as 1 week of age, long before the accumulation of a surrogate UPS substrate becomes discernible. The increase in autophagosomes is confirmed by probing with a transgenic GFP-LC3 and by electron microscopy. Autophagic flux assessments reveal that the removal of autophagosomes in cardiomyocytes is impaired by Csn8 deficiency and the defective fusion between autophagosomes and lysosomes may be responsible. Rab7 transcript and protein levels in the heart are significantly decreased by Csn8 deficiency. Confocal microscopy reveals a striking reciprocal relationship between increases in GFP-LC3 puncta and the decreased Rab7 expression. Rab7 knockdown impairs the removal of autophagosomes in cultured cardiomyocytes. Hence, Csn8/CSN is a central regulator in not only the UPS but also autophagy. Csn8/CSN supports autophagosome-lysosome fusion likely by stimulating Rab7 expression.

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COP9 Signalosome Suppresses RIPK1-RIPK3–Mediated Cardiomyocyte Necroptosis in Mice

Circulation Heart Failure ◽

10.1161/circheartfailure.120.006996 ◽

2020 ◽

Vol 13 (8) ◽

Cited By ~ 3

Author(s):

Peng Xiao ◽

Changhua Wang ◽

Jie Li ◽

Huabo Su ◽

Liuqing Yang ◽

...

Keyword(s):

Heart Failure ◽

Kinase Inhibitor ◽

Mitochondrial Permeability Transition ◽

Ubiquitin Proteasome System ◽

Cop9 Signalosome ◽

Protein Carbonyls ◽

Blue Dye ◽

Cyclophilin D ◽

Littermate Control ◽

Ubiquitin Proteasome

Background: Mechanisms governing the induction of heart failure by the impairment of autophagy and the ubiquitin-proteasome system and the molecular pathways to cardiomyocyte necrosis remain incompletely understood. COPS8 is an essential subunit of the COP9 (COnstitutive Photomorphogenesis 9) signalosome, a key regulator of ubiquitination. Mice with cardiomyocyte-restricted knockout of Cops8 (Cops8-cko) show autophagic and ubiquitin-proteasome system malfunction and massive cardiomyocyte necrosis followed by acute heart failure and premature death, providing an excellent animal model to address the mechanistic gaps specified above. This study was conducted to determine the nature and underlying mechanisms of the cardiomyocyte necrosis in Cops8-cko mice. Methods and Results: Compared with littermate control mice, myocardial protein levels of key factors in the necroptotic pathway (RIPK1 [receptor-interacting protein kinase 1], RIPK3, MLKL [mixed lineage kinase-like], the RIPK1-bound RIPK3), protein carbonyls, full-length Casp8 (caspase 8), and BCL2, as well as histochemical staining of superoxide anions were significantly higher but the cleaved Casp8 and the Casp8 activity were significantly lower in Cops8-cko mice. In vivo cardiomyocyte uptake of Evan’s blue dye was used as an indicator of necrosis. Cops8-cko mice treated with a RIPK1 kinase inhibitor (Nec-1 [Necrostatin-1]) showed less Evans blue dye uptake (0.005% versus 0.20%; P <0.0001) and longer median lifespan (32.5 versus 27 days; P <0.01) than those treated with vehicle control. RIPK3 haploinsufficiency showed similar rescuing effects on Cops8-cko but Cyclophilin D deficiency did the opposite. Conclusions: Cardiac Cops8/COP9 signalosome malfunction causes RIPK1-RIPK3 dependent, but mitochondrial permeability transition pore independent, cardiomyocyte necroptosis in mice and the COP9 signalosome plays an indispensable role in suppressing cardiomyocyte necroptosis.

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ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization

International Journal of Molecular Sciences ◽

10.3390/ijms22052689 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2689

Author(s):

Jianmin Si ◽

Chris Van den Haute ◽

Evy Lobbestael ◽

Shaun Martin ◽

Sarah van Veen ◽

...

Keyword(s):

Membrane Integrity ◽

Ubiquitin Proteasome System ◽

Regulatory Function ◽

Membrane Association ◽

Loss Of Function ◽

Atypical Form ◽

Polyamine Transport ◽

Independent Functions ◽

Ubiquitin Proteasome ◽

The Impact

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson’s disease and Kufor–Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.

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Cullin Deneddylation Suppresses the Necroptotic Pathway in Cardiomyocytes

Frontiers in Physiology ◽

10.3389/fphys.2021.690423 ◽

2021 ◽

Vol 12 ◽

Author(s):

Megan T. Lewno ◽

Taixing Cui ◽

Xuejun Wang

Keyword(s):

Ubiquitin Proteasome System ◽

Arrhythmogenic Right Ventricular Dysplasia ◽

Cop9 Signalosome ◽

E3 Ligases ◽

Protein Subunits ◽

Unique Protein ◽

Recent Advances ◽

Regulated Necrosis ◽

Ubiquitin Proteasome ◽

Apoptosis And Necrosis

Cardiomyocyte death in the form of apoptosis and necrosis represents a major cellular mechanism underlying cardiac pathogenesis. Recent advances in cell death research reveal that not all necrosis is accidental, but rather there are multiple forms of necrosis that are regulated. Necroptosis, the earliest identified regulated necrosis, is perhaps the most studied thus far, and potential links between necroptosis and Cullin-RING ligases (CRLs), the largest family of ubiquitin E3 ligases, have been postulated. Cullin neddylation activates the catalytic dynamic of CRLs; the reverse process, Cullin deneddylation, is performed by the COP9 signalosome holocomplex (CSN) that is formed by eight unique protein subunits, COPS1/CNS1 through COPS8/CNS8. As revealed by cardiomyocyte-restricted knockout of Cops8 (Cops8-cko) in mice, perturbation of Cullin deneddylation in cardiomyocytes impairs not only the functioning of the ubiquitin–proteasome system (UPS) but also the autophagic–lysosomal pathway (ALP). Similar cardiac abnormalities are also observed in Cops6-cko mice; and importantly, loss of the desmosome targeting of COPS6 is recently implicated as a pathogenic factor in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). Cops8-cko causes massive cardiomyocyte death in the form of necrosis rather than apoptosis and rapidly leads to a progressive dilated cardiomyopathy phenotype as well as drastically shortened lifespan in mice. Even a moderate downregulation of Cullin deneddylation as seen in mice with Cops8 hypomorphism exacerbates cardiac proteotoxicity induced by overexpression of misfolded proteins. More recently, it was further demonstrated that cardiomyocyte necrosis caused by Cops8-cko belongs to necroptosis and is mediated by the RIPK1–RIPK3 pathway. This article reviews these recent advances and discusses the potential links between Cullin deneddylation and the necroptotic pathways in hopes of identifying potentially new therapeutic targets for the prevention of cardiomyocyte death.

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