Issue Highlights

[Mini review]

Correlation between retromer and neurological diseases including Alzheimer

Chae-Eun Jang*

Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 46241, Korea

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Current Research Summaries

  • [Mini review] Correlation between retromer and neurological diseases including Alzheimer

    Retromer is an important endosomal trafficking protein assembly that takes part in transporting the membrane cargo. Endosomal trafficking involves sorting of proteins from the endosome and transporting them through different pathways in the cell. The three major pathways of endosomal trafficking are recycling, retrograde, and degradation pathways; retromers are involved in the first two types of pathways. Since 2005, many researches have studied the correlation between retromer and different neurodegenerative diseases. Since the first study on Alzheimer's, numerous studies on other neurodegenerative diseases, including Parkinson’s disease and Neuronal Ceroid Lipofuscinosis, revealed that dysfunction of the retromer contributes to these afflictions. It is therefore important to understand the molecular mechanism behind the process. Recent studies have shown the potential of retromer as a drug target. Therapeutic agents, such as retromer chaperone, have been found to improve the function of retromer without affecting other pathways.

  • [Resource] Facility for high resolution cryo-electron microscopy of biological macromolecules at Korea Basic Science Institute

    Cryo-electron microscopy (cryo-EM) single particle analysis has become a mainstream structural biology tool and therefore access to high-end transmission electron microscope (TEM) dedicated to high resolution cryo-EM is critical. At Korea Basic Science Institute (KBSI), a facility dedicated to biological electron microscopy was established in 2016, and High Resolution Bio-TEM (HR Bio-TEM, Titan Krios, Thermo Fisher Scientific Inc., USA) is now accessible to national and international researchers. This report describes major features of the HR Bio-TEM and the facility, along with overall workflow of cryo-EM image data acquisition.

  • [Crystallization] Crystallization and preliminary X-ray diffraction analysis of a redox-sensing repressor Rex from Thermotoga maritima

    The redox-sensing repressor Rex is a homodimeric transcriptional regulator involved in expression of respiratory genes. Because nicotinamide adenine dinucleotide (NAD) exists oxidized or reduced form by catabolic metabolism, intracellular NAD+/NADH ratio can be a key signal indicating the cellular redox state. The Rex from hyperthermophilic bacterium, Thermotoga maritima (TmRex), was cloned and overexpressed in Escherichia coli. The TmRex is composed of 208 amino-acid residues with a molecular mass of 22,954 Da. The TmRex crystals were obtained by the sitting-drop vapourdiffusion method and diffracted to 1.95 Å resolution. The crystals belonged to the monoclinic space group P21, with unitcell parameters a = 53.54 Å, b = 88.34 Å, c = 87.84 Å, and β = 96.74°. Two dimeric molecules of TmRex were present in an asymmetric unit, giving a solvent contents of 45.17%.

  • [Crystallization] Phase determination of a homogentisate dioxygenase from Comamonas sp. strain P19

    In Comamonas sp. strain P19, homogentisate 1,2-dioxygenase (HGO) catalyzes the conversion of homogentisate to 4-maleylacetoacetate by aromatic ring scission, in the breakdown of tyrosine and phenylalanine. To determine the molecular background of the enzymatic mechanism of HGO in this zinc-resistant organism, hmgA encoding HGO of Comamonas sp. strain P19 was cloned, and the expressed protein was purified. The protein was crystallized in solutions I [25% (w/v) polyethylene glycol 3350 and 0.1 M trisodium citrate at pH 5.6] and II [1.4 M ammonium tartrate and 0.1 M bis- Tris at pH 5.5]. X-ray diffraction data were collected to 1.8 Å resolution using synchrotron radiation. The crystal belongs to the orthorhombic space group P212121, with unit cell dimensions of a = 73.2 Å, b = 100.0 Å, and c = 134.9 Å. A traceable electron density map was calculated using anomalous diffraction data obtained from a crystal soaked in zinc ions.

  • [Crystallization] Crystallization and preliminary structural study of the Thermobaculum terrenum Csh2 in the type IB CRISPR system

    CRISPR/Cas system is an RNA-mediated prokaryotic adaptive immune system against mobile genetic elements. Among three CRISPR/Cas systems, type I system has two functional units in removing nucleic acids, Cascade and Cas3. Type IB Cascade has been analyzed to have components of Csh1, Csh2, Cas4, and Cas5, whose structural and functional information is not available. To provide a structural background for the type IB Cascade, the preliminary structural study of the Csh2 from Thermobaculum terrenum is reported here. The purified recombinant protein was successfully crystallized using the precipitant solution composed of 10% (w/v) Polyethylene Glycol 3350, 2% (v/v) Tacsimate at pH 5.0, and 100 mM Sodium Citrate tribasic dihydrate at pH 5.6. Diffraction data were collected to a resolution of 1.93 Å using synchrotron radiation. The crystal belongs to the primitive triclinic P1 space group, with unit cell dimensions a = 31.04 Å, b = 54.65 Å, c = 96.46 Å, α = 73.6 º, β = 88.4 º and γ = 89.8 º. With two molecules in the asymmetric unit, the crystal volume per unit protein weight corresponds with 2.29 Å3·Da-1, resulting in 46% solvent.

  • [Crystallization] Purification, crystallization, and X-ray crystallographic analysis of Vac8p complexed with Atg13p from Saccharomyces cerevisiae

    Vac8p is a vacuolar protein that plays pivotal roles in both vacuole inheritance and the formation of nucleus vacuole junction (NVJ) in yeast. The Vac8p directly interacts with Atg13p, a component of the autophagy machinery, and mediates cytoplasm-to-vacuole targeting (Cvt) pathway, resulting in the maturation of aminopeptidase I (Ape1p). Here, we coexpressed and purified Saccharomyces cerevisiae Vac8p complexed with Atg13p in Escherichia coli bacteria cells, and crystallized the complex proteins under the condition of 25% (v/v) PEG 400, 100 mM Tris pH 8.5, 2% (v/v) Ethylene glycol, 2% (w/v) PEG 3350, 1.5% (w/v) PEG 20000, 5 mM DTT at 293K. X-ray diffraction data of the crystals were collected to 2.9 Å resolution at the synchrotron radiation. The crystals belong to the orthorhombic space group P212121 with unit cell parameters a = 62.7 Å, b = 92.4 Å, and c = 139.9 Å. The asymmetric unit contains one Vac8p-Atg13p heterodimer with a corresponding VM of 2.92 Å3 Da-1 and solvent content of 57.8%.

  • [Crystallization] Purification, crystallization and X-ray crystallographic analysis of the type VI secretion system accessory protein TagF from Pseudomonas aeruginosa

    TagF is the product of the type VI secretion system (T6SS) accessory gene F, which is capable of regulating T6SS assembly in Pseudomonas aeruginosa. Experimental results suggest that TagF may post-translationally regulate the T6SS through phosphatase activity, but the physiological functions of TagF are not yet understood. To provide structural insight into TagF function and the regulatory mechanisms controlling the T6SS, N-terminally His6-tagged TagF was overexpressed, purified, and crystallized using hanging-drop vapor diffusion in a solution of 2.5 M NaCl and 0.1 M Bis-Tris propane (pH 7.0). X-ray diffraction data from TagF crystals was collected at a resolution of 2.7 Å. TagF crystals belonged to the space group P21212, with unit cell parameters a = 93.7, b = 92.4, c = 151.1 Å, and α = β = γ = 90°. TagF consists of four independent subunits in the asymmetric unit.

  • [Crystallization] Purification, crystallization and X-ray diffraction of heparan sulfate bounded human RAGE

    Receptor for advanced glycation end products (RAGE) is one of the single transmembrane domain containing receptors and causes various inflammatory diseases including diabetes and atherosclerosis. RAGE extracellular domain has three consecutive IgG-like domains (V-C1-C2 domain) which interact with various soluble ligands including heparan sulfate or HMGB1. Studies have shown that each ligand induces different oligomeric forms of RAGE which results in a ligandspecific signal transduction. The structure of mouse RAGE bound to heparan sulfate has been previously determined but the electron density map of heparan sulfate was too ambiguous that the exact position of heparin sulfate could not be defined. Furthermore, the complex structure of human RAGE and heparin sulfate still remains elusive. Therefore, to determine the structure, human RAGE was overexpressed using bacterial expression system and crystallized using the sitting drop method in the condition of 0.1 M sodium acetate trihydrate pH 4.6, 8 % (w/v) polyethylene glycol 4,000 at 290 K. The crystal diffracted to 3.6 Å resolution and the space group is C121 with unit cell parameters a= 206.04 Å, b= 68.64 Å, c= 98.73 Å, α= 90.00°, β= 90.62°, γ= 90.00°.