HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress

Zhang, Liyong; Chen, Xin; Sharma, Parveen; Moon, Mark; Sheftel, Alex D.; Dawood, Fayez; Nghiem, Mai; Wu, Jun; Li, Ren‐Ke; Gramolini, Anthony O.; Sorensen, Poul H.; Penninger, Josef; Brumell, John H.

doi:10.1038/ncomms4430

Cited by 35 publications

(32 citation statements)

References 48 publications

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“…It is generally agreed that ubiquitin conjugation to substrates drives the retro‐translocation process and transfers the ubiquitinated substrates to the cytosolic 26S‐proteasome for digestion. Thus, levels of ubiquitin‐conjugated proteins can reflect the activity of degradation (Su and Wang, ; Zhang et al , ). As shown in Figure A and Supporting Information Fig.…”

Section: Resultsmentioning

confidence: 99%

Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol‐requiring enzyme 1α/X box‐binding protein‐1 pathway

Wang

Wang²,

Fan

et al. 2016

British J Pharmacology

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*These two authors contributed equally to this work. BACKGROUND AND PURPOSEEndoplasmic reticulum (ER) stress is increasingly recognized as an important causal factor of many diseases. Targeting ER stress has now emerged as a new therapeutic strategy for treating cardiovascular diseases. Here, we investigated the effects and underlying mechanism of ginkgolide K (1,10-dihydroxy-3,14-didehydroginkgolide, GK) on cardiac ER stress. EXPERIMENTAL APPROACHCell death, apoptosis and ER stress-related signalling pathways were measured in cultured neonatal rat cardiomyocytes, treated with the ER stress inducers tunicamycin, hydrogen peroxide and thapsigargin. Acute myocardial infarction was established using left coronary artery occlusion in mice, and infarct size was measured by triphenyltetrazolium chloride staining. Echocardiography was used to assess heart function and transmission electron microscopy for evaluating ER expansion. KEY RESULTSGinkgolide K (GK) significantly decreased ER stress-induced cell death in both in vitro and in vivo models. In ischaemic injured mice, GK treatment reduced infarct size, rescued heart dysfunction and ameliorated ER dilation. Mechanistic studies revealed that the beneficial effects of GK occurred through enhancement of inositol-requiring enzyme 1α (IRE1α)/X box-binding protein-1 (XBP1) activity, which in turn led to increased ER-associated degradation-mediated clearance of misfolded proteins and autophagy. In addition, GK was also able to partly repress the pro-apoptotic action of regulated IRE1-dependent decay and JNK pathway. CONCLUSIONS AND IMPLICATIONSIn conclusion, GK acts through selective activation of the IRE1α/XBP1 pathway to limit ER stress injury. GK is revealed as a promising therapeutic agent to ameliorate ER stress for treating cardiovascular diseases. AbbreviationsASK1, apoptotic signalling kinase-1; ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; ERAD, ER-associated degradation; eIF2α, eukaryotic translation initiation factor 2α; GB, ginkgolide B; GK, ginkgolide K; GRP78, glucose-regulated protein 78 kD; IRE1α, inositol-requiring enzyme 1α; NRCMs, neonatal rat cardiomyocytes; PERK, pancreatic eIF2α kinase; RIDD, regulated IRE1-dependent decay; TRAF2, TNF receptor-associated factor 2; UPR, unfolded protein response; XBP1, X box-binding protein-1 BJP IntroductionThe endoplasmic reticulum (ER) is a membranous organelle that plays an important role in the maintenance of cellular processes such as protein processing, calcium homeostasis and lipid biosynthesis. Effective ER function is essential for the folding of secretory and membrane proteins (Wei and Hendershot, 1996). A number of cellular stresses disturb the balance between the folding capacity of the ER and the burden of incoming proteins, including oxidative stress, ischaemic insult, abnormal ER calcium content, and enhanced expression of normal and/or folding-defective proteins. These events lead to the accumulation of unfo...

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Section: Resultsmentioning

confidence: 99%

Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol‐requiring enzyme 1α/X box‐binding protein‐1 pathway

Wang

Wang²,

Fan

et al. 2016

British J Pharmacology

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“…Recent studies by Zhang et al (22) and Rotblat et al (28) showed that a homolog to the E6-AP carboxyl terminus domain and HACE1 is required for modulating stress responses in the heart and brain via autophagy. Deleting the Hace1 gene resulted in reduced autophagy flux in a p62-dependent manner in a severe transaortic constricted (sTAC) mouse heart, suggesting that HACE1 is critical in mediating p62-dependent autophagy flux in myocytes (22). We investigated PLN levels in Hace1-null mice.…”

Section: Resultsmentioning

confidence: 99%

Metformin increases degradation of phospholamban via autophagy in cardiomyocytes

Teng

Miyake

Yokoe

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Phospholamban (PLN) is an effective inhibitor of the sarco(endo) plasmic reticulum Ca 2+ ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [ 35 S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.selective autophagy | ubiquitinylation | protein degradation P hospholamban (PLN) is a 52-amino acid peptide located in the sarcoplasmic reticulum (SR) membrane in cardiac, slowtwitch skeletal, and smooth muscle, where it exists as a monomer or pentamer. Whereas monomeric PLN physically interacts with sarco(endo)plasmic reticulum Ca 2+ ATPase type 2a (SERCA2a) to antagonize its function, pentameric PLN complexes are thought to be a reservoir of inactive PLN (1-3). The physical interaction between SERCA2a and PLN reduces the apparent affinity of SERCA2a for Ca 2+ , thereby making SERCA2a less active in transporting Ca 2+ from the cytoplasm to the lumen of the SR at the same concentration of cytoplasmic Ca 2+. The physical interaction between the two proteins is regulated by phosphorylation of PLN at Ser16 by protein kinase A or at Thr17 by Ca 2+ /calmodulin-dependent protein kinase II (2). Phosphorylation of PLN reduces its affinity for SERCA2a, thereby increasing SERCA2a activity (2). Evidence from transgenic mice also supports the inhibitory function of PLN. Although targeted PLN deletion enhances baseline cardiac performance, cardiac-specific overexpression of superinhibitory forms of PLN leads to decreases in the affinity of SERCA2a for Ca 2+ (2). These observations underscore the primary role of PLN as a regulator of SERCA2a activity and, therefore, as a crucial regulator of cardiac contractility. PLN inhibition of SERCA2a can be reversed by either external (i.e., activation of β-adrenergic receptors) or internal (i.e., increased...

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“…An important component in maintaining proteostasis is the rapid digestion of mis-folded species. While autophagy plays a role in this process, particularly under certain pathological conditions such as heart failure (3), the bulk of this “janitorial function” in healthy cells is mediated by the proteasome (4). Low levels of proteasome activity exacerbate protein-folding diseases, while higher proteasome activity ameliorate them (5–14).…”

Section: Introductionmentioning

confidence: 99%

Establishment of a suite of assays that support the discovery of proteasome stimulators

Trader

Simanski

Dickson

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background The proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing. Methods The cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators. We have developed follow-on assays that employ more physiologically relevant substrates. Results To demonstrate the efficacy of this workflow, the NIH Clinical Collection (NCC) was screened. While many compounds stimulated proteasome-mediated proteolysis of the pro-fluorogenic peptide substrates, most failed to evince activity in assays with larger peptide or protein substrates. We also show that two molecules claimed previously to be proteasome agonists, oleuropein and betulinic acid, indeed accelerate hydrolysis of the fluorogenic substrate, but have no effect on the turnover of a mis-folded protein in vitro or in cellulo. However, two small molecules from the NCC, MK-866 and AM-404, stimulate the proteasome-mediated turnover of a mis-folded protein in living cells by 3- to 4-fold. Conclusion Assays that monitor the proteasome-mediated degradation of larger peptides and proteins can distinguish bona fide agonists from compounds only able to stimulate the cleavage of short, non-physiologically relevant peptides. General significance A suite of assays has been established that allows the discovery of bona fide proteasome agonists. AM-404 and MK-866 can be useful tools for cell culture experiments, and can serve as scaffolds to generate more potent 20S stimulators.

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HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress

Cited by 35 publications

References 48 publications

Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol‐requiring enzyme 1α/X box‐binding protein‐1 pathway

Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol‐requiring enzyme 1α/X box‐binding protein‐1 pathway

Metformin increases degradation of phospholamban via autophagy in cardiomyocytes

Establishment of a suite of assays that support the discovery of proteasome stimulators

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