EL's+Logbook+week+of+Jan+21

Faculty meeting on Tuesday, Which means, we did skip school for two days in a row! ||  ||
 * 01/21/08~01/22/08**
 * ===Information=== ||
 * ===What I did & Opinion=== ||
 * Marting Luther King Jr. Day on Monday,
 * Marting Luther King Jr. Day on Monday,


 * 01/23/08**
 * ===Information=== ||
 * [|**http://www.pnas.org/cgi/reprint/0437840100v1.pdf**]
 * Article>> Structure of a Cholesterol-Binding Protein Deficient in Niemann-Pick Type C2 Disease by Natalia Friedland, Heng-Ling Liou, Peter Lobel, and ann M. Stock** ||  ||

information about NPC2 protein. ||  ||
 * ===What I did & Opinion=== ||
 * I printed out the article linked above the column, and I started to read article which is about 6 pages long. I will pinpoint some important contents in the article, and list new

01/24/08 The neurodegenerative disease Niemann–Pick Type C2 (NPC2) results from mutations in the //NPC2// (//HE1//) gene that cause abnormally high cholesterol accumulation in cells. We find that purified NPC2, a secreted soluble protein, binds cholesterol specifically with a much higher affinity (//K//d 5 30–50 nM) than previously reported. Genetic and biochemical studies identified single amino acid changes that prevent both cholesterol binding and the restoration of normal cholesterol levels in mutant cells. The amino acids that affect cholesterol binding surround a hydrophobic pocket in the NPC2 protein structure, identifying a candidate sterol-binding location. On the basis of evolutionary analysis and mutagenesis, three other regions of the NPC2 protein emerged as important, including one required for efficient secretion.
 * ===Information=== ||
 * [|****http://www.pnas.org/cgi/reprint/0530027100v1.pdf****]

Niemann–Pick Type C (NPC) is an autosomal-recessive disorder characterized by progressive ataxia, dystonia, and dementia (1). Substantial cell death, particularly in the cerebellum, accounts for some of the symptoms. At the cellular level,mutant cells accumulate cholesterol and other lipids in aberrant compartments with features of late endosomes and lysosomes, and the normal homeostatic response to this excess cholesterol is abolished (2–4).Our understanding of the molecular basis of this disease has progressed substantially over the last several years, because the two genes damaged by NPC mutations, //NPC1// and //NPC2//, have been identified (5–7). //NPC1// encodes a multiple-membranespanning protein containing sequences similar to the sterol cleavage activating protein (SCAP) regulator of cholesterol metabolism and the receptor Patched, a transducer of the Hedgehog family of protein signals. Mutations in //NPC1// are responsible for '95% of the NPC cases (8). The exact role that NPC1 plays in maintaining normal levels of cholesterol in late endosomes and lysosomes remains mysterious. Even less is known about the NPC2 [human epididymis (HE1)] protein, the loss of which is responsible for 5% of NPC cases. NPC2 is a small secreted glycoprotein originally identified as a transcript enriched in the HE1 (9). The porcine HE1 homolog was reported to bind cholesterol with micromolar affinity ( //K//d 52.3 mM; ref. 10), information that was crucial in identifying HE1has a candidate //npc2// gene product (7). However, the similarity between the reported //K//d and the solubility of cholesterol in aqueous solution (5 mM, Merck Index) warrants reexamination of the strength and specificity of this interaction. If the binding is meaningful, NPC2 activity could be altered when cholesterol is bound, thus serving as a sensor of cholesterol levels, accessibility, or location. Alternatively, NPC2 could process or transport cholesterol. Resolving these questions is crucial for determining why cholesterol accumulates in people lacking functional NPC2 protein and for designing rational treatment strategies.In this report, we combine //in vitro// binding experiments, genetic analysis, a quantitative cell-based assay of NPC2 function, and sequence evolution information to investigate how NPC2 works. In the accompanying paper by Friedland //et al.// (11), the crystal structure of NPC2 is reported, further revealing the unique properties of the proposed cholesterol-binding site.

Materials and Methods Plasmids. Full-length mouse NPC2 cDNA was amplified from Clone 408396 (Research Genetics, Huntsville, AL). The PCR product was cloned into the //Eco//RI and //Xho//I sites in pcDNA3 (Invitrogen) to yield pNPC2. For the myc 6xHis-tagged construct, the PCR product was inserted into the //Eco//RI and //Kpn//I sites in pcDNA 3.1(2)ymyc-his A (Invitrogen) to yield pNPC2myc-his. In both cases, the insert was fully sequenced. To generate stably expressing cells, a bicistronic construct was made by inserting the 5.2-kb //Xba//Iy//Afl//II fragment from pNPC2myc-his into pIRESpuro2 (CLONTECH) digested with //Nhe//I and //Afl//II to yield pNPC2myc-his-puro. Point mutants were generated by using QuikChange (Stratagene) according to the manufacturer’s instructions.

Cell Culture and Establishment of Chinese Hamster Ovary (CHO)-KI Cells Stably Expressing NPC2-myc-his.** CHO-KI cells (American Type Culture Collection) were grown in F12 (10% FBSy10mg/ml gentamicin). Human NPC2 fibroblasts were the kind gift of Dan Ory (Washington University, St. Louis) and were grown in DMEM (10% FBSy1 mM sodium pyruvatey0.1 mM nonessential amino acidsy2 mM glutamineypenicillinystreptomycin). For experiments involving lipid-free media, cells were washed three times and then incubated in DMEM (1% BSA y0.1 mM nonessential amino acidsy2 mM glutamineypenicillinystreptomycin). For the generation of stably expressing NPC2-myc-his cells, CHO-KI cells were transfected with pNPC2myc-his-puro by using Fugene6 (Roche Molecular Biochemicals). Forty-eight hours posttransfection, the cells were passaged, and transformants were selected with 12 mgyml puromycin. After 2 weeks of selection, colonies were picked with cloning rings. The conditioned medium from each isolated colony was screened for NPC2-myc-his production level by protein blotting with the anti-myc 9E10 antibody (Sigma). For detection of intracellular NPC2-myc-his, a polyclonal NPC2 antibody (a gift from Peter Lobel, Robert Wood Johnson Medical School, Piscataway, NJ) was used to avoid problems due to degradation of the myc-his tag. ||  ||
 * ===What I did & Opinion=== ||