Young Mi Lee

Association of human kinesin superfamily protein member 4 with BRCA2-associated factor 35.

A large portion of human kinesin superfamily protein member 4 (KIF4) is associated with the nuclear matrix during the interphase, while a small portion is found in the cytoplasm. During mitosis, it is associated with chromosomes throughout the entire process. In the present study, we identified a protein that interacts with KIF4 using a yeast two-hybrid system, co-immunoprecipitation and co-fractionation. This protein is BRCA2-associated factor 35 (BRAF35) containing a non-specific DNA binding high-mobility-group domain and a kinesin-like coiled-coil domain. It appeared that the interaction between the two proteins occurs through their respective alpha-helical coiled-coil domains. The co-fractionation experiment revealed that KIF4 and BRAF35 were present in a complex of approx. 540 kDa. The composition and biological significance of this complex should be studied further.

Cell cycle-regulated expression and subcellular localization of a kinesin-8 member human KIF18B

The human genome contains genes encoding for over 40 different types of kinesin and kinesin-like proteins. Of these, the functions of 13 kinesins remain uncharacterized. In this study, we constructed a plasmid containing the ORF of KIF18B and revealed that the KIF18B message of approximately 3kb is expressed in a tissue- and cell type-specific manners. A polypeptide of 842 amino acids was deduced from the ORF sequence. We identified another form of 873 amino acids which arises from alternative splicing at the C-terminal end. We also generated an anti-KIF18B antibody which detects a protein band of 120kDa. Western analyses showed that the protein level of KIF18B is elevated at late G(2) through metaphase, very similar to cyclin B1. Immunocytochemical staining revealed that the KIF18B protein is present predominantly in the nucleus and to a lesser extent in the cytoplasm of interphase cells. During mitosis, most KIF18B was found to be closely associated with astral microtubules emanating from the spindle pole during prometaphase and metaphase. Meanwhile, KIF18B was not detected at anaphase and telophase, consistent with the Western blotting data. The nuclear localization signal was roughly determined by using several EGFP-tagged deletion mutants of KIF18B. Together, the expression of KIF18B is regulated in a cell cycle-dependent manner and therefore may play an important role(s) in cell division.

Overexpression of OLE1 enhances stress tolerance and constitutively activates the MAPK HOG pathway in Saccharomyces cerevisiae

OLE1 of Saccharomyces cerevisiae encodes the sole and essential Δ‐9 desaturase catalyzing the conversion of saturated to unsaturated fatty acids. Upon ectopic overexpression of OLE1 in S. cerevisiae, significant increases in the membrane oleic acid content were observed. OLE1‐overexpressing strains displayed enhanced tolerance to various stresses, better proton efflux, lower membrane permeability, and lessened internal hydrogen peroxide content. The OLE1‐mediated enhanced stress tolerance was considerably diminished upon deletion of HOG1, which encodes the mitogen‐activated protein kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway. Furthermore, OLE1 overexpression constitutively activated Hog1, which remained in the cytoplasm. Hog1 activation was accomplished through the MAPK kinase kinase (MAPKKK) Ssk2, but not Ste11 and Ssk22, the other MAPKKKs of the HOG pathway. Despite its cytoplasmic location, activated Hog1 was able to activate the expression of its canonical targets, including CTT1, HSP12, and STL1, and further, the cAMP and stress response elements present in the promoter. OLE1 overexpression neither caused nor relieved endoplasmic reticulum stress. Individually or in combination, the physiological and molecular changes caused by OLE1 overexpression may contribute to enhanced tolerance to various types of stress. Biotechnol. Bioeng. 2017;114: 620–631.

Dissection of the HOG pathway activated by hydrogen peroxide in Saccharomyces cerevisiae

Cells usually cope with oxidative stress by activating signal transduction pathways. In the budding yeast Sacchromyces cerevisiae, the high osmolarity glycerol (HOG) pathway has long been implicated in transducing the oxidative stress-induced signal, but the underlying mechanisms are not well defined. Based on phosphorylation of the mitogen-activated protein kinase (MAPK) Hog1, we reveal that the signal from hydrogen peroxide (H2 O2 ) flows through Ssk1, the response regulator of the two-component system of the HOG pathway. Downstream signal transduction into the HOG MAPK cascade requires the MAP kinase kinase kinase (MAP3K) Ssk2 but not its paralog Ssk22 or another MAP3K Ste11 of the pathway, culminating in Hog1 phosphorylation via the MAP2K Pbs2. When overexpressed, Ssk2 is also activated in an Ssk1-independent manner. Unlike in mammals, H2 O2 does not cause endoplasmic reticulum stress, which can activate Hog1 through the conventional unfolded protein response. Hog1 activated by H2 O2 is retained in the cytoplasm, but is still able to activate the cAMP- or stress-responsive elements by unknown mechanisms.

Characterization of human SMARCE1r high-mobility-group protein.

The high-mobility-group (HMG) proteins are chromatin-associated proteins that are common to all higher organisms. They bind DNA in a sequence-specific or non-sequence-specific way to induce DNA bending, and regulate chromatin function and gene expression. Here we report the characterization of an HMG box-containing gene, designated human Smarce1r gene. It contained an open reading frame (ORF) encoding 317 amino acids and had 86% and 94% identity with the murine Smarce1r ORF at the nucleic acid and amino acid level, respectively. A putative nuclear localization signal, one HMG domain, and a coiled-coil domain were localized. A single transcript of 1.6 kb was ubiquitously expressed in various human tissues except for the fetal brain in which the transcript was barely detected. Western blot analysis revealed that human SMARCE1r was expressed in specific tissues such as colon and placenta. Subcellular fractionation, DNA-affinity column chromatography, and electrophoretic mobility shift assays showed that human SMARCE1r was associated with the nuclear matrix and that it possessed DNA binding activity, as expected.

Association between Metformin Use and Risk of Lactic Acidosis or Elevated Lactate Concentration in Type 2 Diabetes

Purpose Metformin can reduce diabetes-related complications and mortality. However, its use is limited because of potential lactic acidosis-associated adverse effects, particularly in renal impairment patients. We aimed to investigate the association of metformin use with lactic acidosis and hyperlactatemia in patients with type 2 diabetes. Materials and Methods This was a cross-sectional study from a tertiary university-affiliated medical center. A total of 1954 type 2 diabetes patients were recruited in 2007–2011, and stratified according to the estimated glomerular filtration rate of 60 mL/min/1.73 m2. Lactic acidosis was defined as plasma lactate levels >5 mmol/L and arterial pH <7.35. Results Metformin was used in 61.4% of the patients with type 2 diabetes mellitus. Plasma lactate levels were not different in the patients with and without metformin use. There was no difference in prevalence of hyperlactatemia and lactic acidosis between the patients with and without metformin use (18.9% vs. 18.7%, p=0.905 for hyperlactatemia and 2.8% vs. 3.3%, p=0.544 for lactic acidosis). Similar results were observed in the patients with estimated glomerular filtration rate <60 mL/min/1.73 m2. Most patients with lactic acidosis had at least one condition related to hypoxia or poor tissue perfusion. Multiple regression analysis indicated no association between metformin use and lactic acidosis, whereas tissue hypoxia was an independent risk factor for lactic acidosis [odds ratio 4.603 (95% confidence interval, 1.327–15.965)]. Conclusion Metformin use was not associated with hyperlactatemia or lactic acidosis in patients with type 2 diabetes.