Eunmin Kim

Herp enhances ER-associated protein degradation by recruiting ubiquilins

ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3delta, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3delta was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates.

Genetic analysis of the cardiac sodium channel gene SCN5A in Koreans with Brugada syndrome.

AbstractThe SCN5A gene encodes the alpha subunit of the human cardiac voltage-gated sodium channel. Mutations in SCN5A are responsible for Brugada syndrome, an inherited cardiac disease that leads to idiopathic ventricular fibrillation (IVF) and sudden death. In this study, we screened nine individuals from a single family and 12 sporadic patients who were clinically diagnosed with Brugada syndrome. Using PCR-SSCP, DHPLC, and DNA sequencing analysis, we identified a novel single missense mutation associated with Brugada syndrome in the family and detected a C5607T polymorphism in Korean subjects. A single nucleotide substitution of G to A at nucleotide position 3934 changed the coding sense of exon 21 of the SCN5A from glycine to serine (G1262S) in segment 2 of domain III (DIII-S2). Four individuals in the family carried the identical mutation in the SCN5A gene, but none of the 12 sporadic patients did. This mutation was not found in 150 unrelated normal individuals. This finding is the first report of a novel mutation in SCN5A associated with Brugada syndrome in Koreans.

Tissue-specific expression and subcellular localization of ALADIN, the absence of which causes human triple A syndrome

Triple A syndrome is a rare genetic disorder caused by mutations in the achalasia-addisonianism-alacrima syndrome (AAAS) gene which encodes a tryptophan aspartic acid (WD) repeat-containing protein named alacrima-achalasia-adrenal insufficiency neurologic disorder (ALADIN). Northern blot analysis shows that the 2.1 kb AAAS mRNA is expressed in various tissues with stronger expression in testis and pancreas. We show that human ALADIN is a protein with an apparent molecular weight of 60 kDa, and expressed in the adrenal gland, pituitary gland and pancreas. Furthermore, biochemical analysis using anti-ALADIN antibody supports the previous finding of the localization of ALADIN in the nuclear membrane. The mutations S544G and S544X show that alteration of S544 residue affects correct targeting of ALADIN to the nuclear membrane.

Infarcted Myocardium-Primed Dendritic Cells Improve Remodeling and Cardiac Function After Myocardial Infarction by Modulating the Regulatory T Cell and Macrophage PolarizationClinical Perspective

Background: Inflammatory responses play a critical role in left ventricular remodeling after myocardial infarction (MI). Tolerogenic dendritic cells (tDCs) can modulate immune responses, inducing regulatory T cells in a number of inflammatory diseases. Methods: We generated tDCs by treating bone marrow–derived dendritic cells with tumor necrosis factor-&agr; and cardiac lysate from MI mice. We injected MI mice, induced by a ligation of the left anterior descending coronary artery in C57BL/6 mice, twice with tDCs within 24 hours and at 7 days after the ligation. Results: In vivo cardiac magnetic resonance imaging and ex vivo histology confirmed the beneficial effect on postinfarct left ventricular remodeling in MI mice treated with tDCs. Subcutaneously administered infarct lysate–primed tDCs near the inguinal lymph node migrated to the regional lymph node and induced infarct tissue–specific regulatory T-cell populations in the inguinal and mediastinal lymph nodes, spleen, and infarcted myocardium, indicating that a local injection of tDCs induces a systemic activation of MI-specific regulatory T cells. These events elicited an inflammatory-to-reparative macrophage shift. The altered immune environment in the infarcted heart resulted in a better wound remodeling, preserved left ventricular systolic function after myocardial tissue damage, and improved survival. Conclusions: This study showed that tDC therapy in a preclinical model of MI was potentially translatable into an antiremodeling therapy for ischemic tissue repair.

Genomic organization of the region spanning D14Mit262 and D14Mit86 on mouse chromosome 14 and exclusion of Adam28 and Adamdec1 as the cataract-causing gene, lr2.

Mice with recessive cataract, CXSD, show the first clinical symptoms of cataract at five weeks, with complete penetrance. We previously localized the cataract-causing lens rupture 2 gene (lr2) to mouse chromosome 14. In the process of positional cloning of the lr2 gene, we determined the genomic organization of the critical region, defined by D14Mit262 and D14Mit86, and compared it to recently published map information. In addition, mutational analysis using reverse transcription polymerase chain reaction (RT-PCR) followed by direct sequencing as well as quantitative realtime PCR (RQ-PCR) was performed to investigate Adam28 and Adamdec1 as lr2 candidate genes in this study. There was no mutation cosegregating with the phenotype of CXSD mice, which excluded these genes as the lr2 gene. Identification of more transcripts from this region and their mutation analyses are required to isolate the lr2 gene.

Poly(rC) binding protein 2 acts as a negative regulator of IRES-mediated translation of Hr mRNA

During the hair follicle (HF) cycle, HR protein expression is not concordant with the presence of the Hr mRNA transcript, suggesting an elaborate regulation of Hr gene expression. Here we present evidence that the 5′ untranslated region (UTR) of the Hr gene has internal ribosome entry site (IRES) activity and this activity is regulated by the binding of poly (rC) binding protein 2 (PCBP2) to Hr mRNA. Overexpression and knockdown of PCBP2 resulted in a decrease in Hr 5′ UTR IRES activity and an increase in HR protein expression without changing mRNA levels. We also found that this regulation was disrupted in a mutant Hr 5′ UTR that has a mutation responsible for Marie Unna hereditary hypotrichosis (MUHH) in both mice and humans. These findings suggest that Hr mRNA expression is regulated at the post-transcriptional level via IRES-mediated translation control through interaction with PCPB2, but not in MUHH.