Dukgyu Lee

Calreticulin Induces Dilated Cardiomyopathy

Background Calreticulin, a Ca2+-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart. Methodology/Principal Findings We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca2+ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca2+-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca2+-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin. Conclusions/Significance We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca2+ handling genes, gap junction components and left ventricle remodeling.

UBC9-dependent association between calnexin and protein tyrosine phosphatase 1B (PTP1B) at the endoplasmic reticulum

Background: PTP1B is an enzyme localized to the cytoplasmic face of the ER. Results: Calnexin binds UBC9, is SUMOylated and forms complexes with PTP1B at the ER membrane. Conclusion: This work reveals a previously unrecognized role for calnexin in the retention of PTP1B at the ER membrane. Significance: SUMOylation machinery and UBC9 link two ER proteins from divergent pathways. Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism.

Sumoylation regulates ER stress response by modulating calreticulin gene expression in XBP-1-dependent mode in Caenorhabditis elegans

Excessive accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen causes ER stress, which induces a set of genes, including those encoding ER-resident chaperones, to relieve the detrimental effects and recover homeostasis. Calreticulin is a chaperone that facilitates protein folding in the ER lumen, and its gene expression is induced by ER stress in Caenorhabditis elegans. Sumoylation conjugates small ubiquitin-like modifier (SUMO) proteins with target proteins to regulate a variety of biological processes, such as protein stability, nuclear transport, DNA binding, and gene expression. In this study, we showed that C. elegans X-box-binding protein 1 (Ce-XBP-1), an ER stress response transcription factor, interacts with the SUMO-conjugating enzyme UBC-9 and a SUMOylation target. Our results indicated that abolishing sumoylation enhanced calreticulin expression in an XBP-1-dependent manner, and the resulting increase in calreticulin counteracted ER stress. Furthermore, sumoylation was repressed in C. elegans undergoing ER stress. Finally, RNAi against ubc-9 mainly affected the expression of genes associated with ER functions, such as lipid and organic acid metabolism. Our results suggest that sumoylation plays a regulatory role in ER function by controlling the expression of genes required for ER homeostasis in C. elegans.

Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis.

Background Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR) pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function. Methodology/Principal Findings We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER) homeostasis, transient activation of the unfolded protein response (UPR) pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA) is sufficient to prevent cardiac fibrosis, and improved exercise tolerance. Conclusions We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function.

Calcium and bioenergetics: from endoplasmic reticulum to mitochondria

Abstract Controlling metabolism throughout life is a necessity for living creatures, and perturbation of energy balance elicits disorders such as type-2 diabetes mellitus and cardiovascular disease. Ca2+ plays a key role in regulating energy generation. Ca2+ homeostasis of the endoplasmic reticulum (ER) lumen is maintained through the action of Ca2+ channels and the Ca2+ ATPase pump. Once released from the ER, Ca2+ is taken up by mitochondria where it facilitates energy metabolism. Mitochondrial Ca2+ serves as a key metabolic regulator and determinant of cell fate, necrosis, and/or apoptosis. Here, we focus on Ca2+ transport from the ER to mitochondria, and Ca2+-dependent regulation of mitochondrial energy metabolism.

A screen for genetic loci on the X chromosome required for body‐wall muscle development during embryogenesis in caenorhabditis elegans

We have screened available chromosomal deficiencies on the X chromosome for genetic loci whose zygotic expression is required for body‐wall muscle development during embryogenesis in Caenorhabditis elegans. Previously, it had been reported that no sign of muscle development was detected in nullo‐X embryos arrested at an early stage of embryogenesis. Based on this observation, it has been suggested that genetic loci exist on the X chromosome whose zygotic expression is essential for body‐wall muscle formation. In order to identify such myogenic loci, 9 chromosomal deficiencies covering approximately 45% of the X chromosome have been tested. Homozygous embryos from these deficiency strains were collected and terminal phenotypes of arrested embryos were observed by Nomarski microscopy. As a secondary assay, monoclonal antibodies against two myosin heavy chain (MHC) isoforms, the products of the myo‐3 and unc‐54 genes, were used to detect body‐wall muscle differentiation. All the homozygous deficiency embryos...

Fatty acid binding protein (Fabp) 5 interacts with the calnexin cytoplasmic domain at the endoplasmic reticulum

Calnexin is a type 1 integral endoplasmic reticulum membrane molecular chaperone with an endoplasmic reticulum luminal chaperone domain and a highly conserved C-terminal domain oriented to the cytoplasm. Fabp5 is a cytoplasmic protein that binds long-chain fatty acids and other lipophilic ligands. Using a yeast two-hybrid screen, immunoprecipitation, microscale thermophoresis analysis and cellular fractionation, we discovered that Fabp5 interacts with the calnexin cytoplasmic C-tail domain at the endoplasmic reticulum. These observations identify Fabp5 as a previously unrecognized calnexin binding partner.