Structures of alternatively spliced isoforms of human ketohexokinase

The structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and peripheral KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP have been solved. The differences between KHK-A and KHK-C resulting from the spliced region are subtle and affect thermostability and probably flexibility; the mutations causing fructosuria were modelled

Ketohexokinase: Expression and Localization of the Principal Fructose-metabolizing Enzyme

Ketohexokinase (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and Western blotting of rodent tissues, including those from mouse knockouts, coupled with RT-PCR assays, we determined the distribution of the splice variants. The highly expressed KHK-C isoform localized to hepatocytes in the liver and to the straight segment of the proximal renal tubule. In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of other tissues, and by Western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from experimental artifact. (J Histochem Cytochem 57:763–774, 2009

Immunohistochemical analysis of Tuba8 knockout.

<p><b>A,B.</b> Cerebellum, stained using the Tuba8 monoclonal (Bioserv). (A, control animal; B, knockout animal) Scale bar = 200 μm. Arrow in part A indicates stronger labelling of dendrites in control tissue. <b>C-E.</b> Testis stained using the Tuba8 monoclonal (Bioserv). (C, D, wild-type control, low and higher magnification respectively: C, scale bar = 200 μm; D, scale bar = 100 μm. E, knockout at low magnification. Arrow indicates strong Tuba8 presence peripheral to the nucleus. <b>F,G.</b> Staining for tyrosinated alpha tubulin. Control (F) and knockout (G) testis. Scale bar = 100 μm. <b>H,I.</b> Staining for acetylated alpha tubulin. Control (H) and knockout (I) testis. Scale bar = 100 μm</p

Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as regulator of the innate immune response.

Aicardi-Goutières syndrome is a mendelian mimic of congenital infection and also shows overlap with systemic lupus erythematosus at both a clinical and biochemical level. The recent identification of mutations in TREX1 and genes encoding the RNASEH2 complex and studies of the function of TREX1 in DNA metabolism have defined a previously unknown mechanism for the initiation of autoimmunity by interferon-stimulatory nucleic acid. Here we describe mutations in SAMHD1 as the cause of AGS at the AGS5 locus and present data to show that SAMHD1 may act as a negative regulator of the cell-intrinsic antiviral response.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe