House of Bread Case Study

Sheila McCann became bored with her public defender job and decided to open her own business. She made a list what she wanted from a business and came to open House of Bread. She uses high quality ingredients and stresses the craft nature of her small batch baking. With her San Luis Obispo store running well, she looked for where else she would like to live and settled on Truckee, CA, known for its outdoor activities. With this new store she is exploring franchising

Analysis of Jmjd6 Cellular Localization and Testing for Its Involvement in Histone Demethylation

BACKGROUND: Methylation of residues in histone tails is part of a network that regulates gene expression. JmjC domain containing proteins catalyze the oxidative removal of methyl groups on histone lysine residues. Here, we report studies to test the involvement of Jumonji domain-containing protein 6 (Jmjd6) in histone lysine demethylation. Jmjd6 has recently been shown to hydroxylate RNA splicing factors and is known to be essential for the differentiation of multiple tissues and cells during embryogenesis. However, there have been conflicting reports as to whether Jmjd6 is a histone-modifying enzyme. METHODOLOGY/PRINCIPAL FINDINGS: Immunolocalization studies reveal that Jmjd6 is distributed throughout the nucleoplasm outside of regions containing heterochromatic DNA, with occasional localization in nucleoli. During mitosis, Jmjd6 is excluded from the nucleus and reappears in the telophase of the cell cycle. Western blot analyses confirmed that Jmjd6 forms homo-multimers of different molecular weights in the nucleus and cytoplasm. A comparison of mono-, di-, and tri-methylation states of H3K4, H3K9, H3K27, H3K36, and H4K20 histone residues in wildtype and Jmjd6-knockout cells indicate that Jmjd6 is not involved in the demethylation of these histone lysine residues. This is further supported by overexpression of enzymatically active and inactive forms of Jmjd6 and subsequent analysis of histone methylation patterns by immunocytochemistry and western blot analysis. Finally, treatment of cells with RNase A and DNase I indicate that Jmjd6 may preferentially associate with RNA/RNA complexes and less likely with chromatin. CONCLUSIONS/SIGNIFICANCE: Taken together, our results provide further evidence that Jmjd6 is unlikely to be involved in histone lysine demethylation. We confirmed that Jmjd6 forms multimers and showed that nuclear localization of the protein involves association with a nucleic acid matrix

Studies on recombinant human 5-aminolaevulinic acid dehydratase and recombinant human porphobilinogen deaminase

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Studies on recombinant human 5-aminolaevulinic acid dehydratase and recombinant human porphobilinogen deaminase

5-Aminolaevulinic acid dehydratase (ALAD) catalyses the condensation between two molecules of 5-aminolaevulinic acid to form porphobilinogen (PBG). Recombinant human ALAD, encoded by a synthetic gene, has been over-expressed and purified to yield milligram amounts of enzyme for characterisation.  The recombinant enzyme was crystallised and the structure revealed a ligand resembling the product, PBG, at the active sites. Experiments described in this thesis indicate the bound ligand is not porphobilinogen but a putative intermediate, which has provided clues to the enzymes mechanism. The human ALAD mutant, Phe12Leu, has been characterised. Phenylalanine 12 is located on the N-terminal arm and is thought to be involved in subunit interactions. Hybrid octamers of the mutant and the normal ALAD were formed, identifying residues that may be important in communication between subunits. Porphobilinogen deaminase (PBGD) polymerises PBG to form the tetrapyrrole preuroporphyrinogen in the third step of the haem biosynthesis pathway. Recombinant erythroid and ubiguitous human PBGD were purified from an engineered strain of E. coli. The purified PBGD appeared homogeneous when analysed by SDS-PAGE but existed in the form of a distinct double band when analysed by non-denaturing PAGE.  Proteolytic digestion and peptide analysis of the two separated deaminase species was carried out to identify differences between the two protein forms. A number of PBGD mutants were studied and some were found to accumulate enzyme substrate (ES) complexes. This thesis describes the purification of these complexes and their attempted crystallisation as the structure of an enzyme substrate complex would provide a wealth of information on the enzyme mechanism.</p

X-Ray Diffraction Images For Human Recombinant 5-Aminolevulinic Acid Dehydratase (Alad).

X-ray diffraction images for recominant human 5-aminolevulinic acid dehydratase (ALAD) collected at ESRF (Grenoble)&nbsp;beam line ID14-2&nbsp;using an ADSC Quantum 4 detector to a resolution of 2.8 &Aring;. A series of 1 &#x30A; oscillation images were&nbsp;recorded with an exposure time of 10 seconds per image. More details are given with the scanned notes and the log file.</span

X-ray diffraction images for human recombinant 5-aminolevulinic acid dehydratase (ALAD).

<p>X-ray diffraction images for recominant human 5-aminolevulinic acid dehydratase (ALAD) collected at ESRF (Grenoble) beam line ID14-2 using an ADSC Quantum 4 detector to a resolution of 2.8 Å. A series of 1 ̊ oscillation images were recorded with an exposure time of 10 seconds per image. More details are given with the scanned notes and the log file. </p

Crystal structure of the 2-Oxoglutarate- and Fe(II)-dependent lysyl hydroxylase JMJD6

Lysyl and prolyl hydroxylations are well-known post-translational modifications to animal and plant proteins with extracellular roles. More recent work has indicated that the hydroxylation of intracellular animal proteins may be common. JMJD6 catalyses the iron- and 2-oxoglutarate-dependent hydroxylation of lysyl residues in arginine-serine-rich domains of RNA splicing-related proteins. We report crystallographic studies on the catalytic domain of JMJD6 in complex with Ni(II) substituting for Fe(II). Together with mutational studies, the structural data suggest how JMJD6 binds its lysyl residues such that it can catalyse C-5 hydroxylation rather than Nepsilon-demethylation, as for analogous enzymes

X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 A) resolution. The cyclic intermediate is bound covalently to Lys(263) with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C-C bond linking both substrates in the intermediate is formed before the C-N bond

Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing

The finding that the metazoan hypoxic response is regulated by oxygen-dependent posttranslational hydroxylations, which regulate the activity and lifetime of hypoxia-inducible factor (HIF), has raised the question of whether other hydroxylases are involved in the regulation of gene expression. We reveal that the splicing factor U2 small nuclear ribonucleoprotein auxiliary factor 65-kilodalton subunit (U2AF65) undergoes posttranslational lysyl-5-hydroxylation catalyzed by the Fe(II) and 2-oxoglutarate–dependent dioxygenase Jumonji domain-6 protein (Jmjd6). Jmjd6 is a nuclear protein that has an important role in vertebrate development and is a human homolog of the HIF asparaginyl-hydroxylase. Jmjd6 is shown to change alternative RNA splicing of some, but not all, of the endogenous and reporter genes, supporting a specific role for Jmjd6 in the regulation of RNA splicing