Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene

Cilia and flagella are microtubule-based structures nucleated by modified centrioles termed basal bodies. These biochemically complex organelles have more than 250 and 150 polypeptides, respectively. To identify the proteins involved in ciliary and basal body biogenesis and function, we undertook a comparative genomics approach that subtracted the nonflagellated proteome of Arabidopsis from the shared proteome of the ciliated/flagellated organisms Chlamydomonas and human. We identified 688 genes that are present exclusively in organisms with flagella and basal bodies and validated these data through a series of in silico, in vitro, and in vivo studies. We then applied this resource to the study of human ciliation disorders and have identified BBS5, a novel gene for Bardet-Biedl syndrome. We show that this novel protein localizes to basal bodies in mouse and C. elegans, is under the regulatory control of daf-19, and is necessary for the generation of both cilia and flagella.     

Expanded CAG repeats activate the DNA damage checkpoint pathway

Trinucleotide repeats (TNRs) are sequences whose expansion causes several genetic diseases and chromosome breakage. We report a novel finding that expanded CAG repeats activate the DNA damage response. Mutations in yeast MEC1, RAD9, or RAD53 genes result in increased rates of fragility of a CAG repeat tract while single or double deletions of RAD17 or RAD24 have only a modest effect on TNR fragility, indicating that signaling down the Rad9 pathway and not the Rad17-Rad24 pathway plays a major role in sensing and repairing CAG-tract breaks. Deletion of CHK1 had no effect on CAG fragility, suggesting that a Chk1-mediated G2 arrest is not required for TNR repair. Absence of Mec1, Ddc2, Rad17, Rad24, or Rad53 also gives rise to increased frequency of CAG repeat contractions, indicating that components of the checkpoint machinery play an active role in the maintenance of both chromosomal integrity and repeat stability at expanded CAG sequences.     

Secretion of recombinant human fibrinogen by the murine mammary gland

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the A, B and fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 g/ml, with total secretion of subunits approaching 700 g/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the B and chains were rate limiting. Both the B and chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for chains over B chains. Also, the subunit complexes ₂, A₂ and the individual subunits A, B and were found as secretion products. When the B was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of B chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.     

Substrate reduction therapy in mouse models of the glycosphingolipidoses

Substrate reduction therapy uses small molecules to slow the rate of glycolipid biosynthesis. One of these drugs, N-butyldeoxynojirimycin (NB-DNJ), shows efficacy in mouse models of Tay-Sachs, Sandhoff and Fabry diseases. This offers the prospect that NB-DNJ may be of therapeutic benefit, at least in the juvenile and adult onset variants of these disorders. The infantile onset variants will require an additional enzyme-augmenting modality if the pathology is to be significantly improved. A second drug, N-butyldeoxyglactonojirimycin, looks very promising for treating storage diseases with neurological involvement as high systemic dosing is achievable without any side-effects.     

The amino acid sequences of the carboxyl termini of human and mouse hepatic lipase influence cell surface association

Human hepatic lipase (hHL) mainly exists cell surface bound, whereas mouse HL (mHL) circulates in the blood stream. Studies have suggested that the carboxyl terminus of HL mediates cell surface binding. We prepared recombinant hHL, mHL, and chimeric proteins (hHLmt and mHLht) in which the carboxyl terminal 70 amino acids of hHL were exchanged with the corresponding sequence from mHL. The hHL, mHL, and hHLmt proteins were catalytically active using triolein and tributyrin as substrates. In transfected cells, the majority of hHLs bound to the cell surface, with only 4% of total extracellular hHL released into heparin-free media, whereas under the same conditions, 61% of total extracellular mHLs were released. Like mHL, hHLmt showed decreased cell surface binding, with 68% of total extracellular hHLmt released. To determine the precise amino acid residues involved in cell surface binding, we prepared a truncated hHL mutant (hHL471) by deleting the carboxyl terminal five residues (KRKIR). The hHL471 also retained hydrolytic activity with triolein and tributyrin, and showed decreased cell surface binding, with 40% of total extracellular protein released into the heparin-free media.These data suggest that the determinants of cell surface binding exist within the carboxyl terminal 70 amino acids of hHL, of which the last five residues play an important role.     

HDL regulates the displacement of hepatic lipase from cell surface proteoglycans and the hydrolysis of VLDL triacylglycerol

We have previously shown that hepatic lipase (HL) is inactive when bound to purified heparan sulfate proteoglycans and can be liberated by HDL and apolipoprotein A-I (apoA-I), but not by LDL or VLDL. In this study, we show that HDL is also able to displace HL directly from the surface of the hepatoma cell line, HepG2, and Chinese hamster ovary cells stably overexpressing human HL. ApoA-I is more efficient at displacing cell surface HL than is HDL, and different HDL classes vary in their ability to displace HL from the cell surface. HDL2s have a greater capacity to remove HL from the cell surface and intracellular compartments, as compared with the smaller HDL particles. The different HDL subclasses also uniquely affect the activity of the enzyme. HDL2 stimulates HL-mediated hydrolysis of VLDL-triacylglycerol, while HDL3 is inhibitory. Inhibition of VLDL hydrolysis appears to result from a decreased interlipoprotein shuttling of HL between VLDL and the smaller, more dense HDL particles. This study suggests that high HDL2 levels are positively related to efficient triacylglycerol hydrolysis by their ability to enhance the liberation of HL into the plasma compartment and by a direct stimulation of VLDL-triacylglycerol hydrolysis.     

Adenovirus E1A,not human papillomavirus E7, sensitizes tumor cells to lysis by macrophages through nitric oxide- and TNF-alpha-dependent mechanisms despite up-regulation of 70-kDa heat shock protein

Expression of adenovirus (Ad) serotype 2 or 5 (Ad2/5) E1A or human papillomavirus (HPV)16 E7 reportedly sensitizes cells to lysis by macrophages. Macrophages possess several mechanisms to kill tumor cells including TNF-alpha, NO, reactive oxygen intermediates (ROI), and Fas ligand (FasL). E1A sensitizes cells to apoptosis by TNF-alpha, and macrophages kill E1A-expressing cells, in part through the elaboration of TNF-alpha. However, E1A also up-regulates the expression of 70-kDa heat shock protein, a protein that inhibits killing by TNF-alpha and NO, thereby protecting cells from lysis by macrophages. Unlike E1A, E7 does not sensitize cells to killing by TNF-alpha, and the effector mechanism(s) used by macrophages to kill E7-expressing cells remain undefined. The purpose of this study was to further define the capacity of and the effector mechanisms used by macrophages to kill tumor cells that express Ad5 E1A or HPV16 E7. We found that Ad5 E1A, but not HPV16 E7, sensitized tumor cells to lysis by macrophages. Using macrophages derived from mice unable to make TNF-alpha, NO, ROI, or FasL, we determined that macrophages used NO, and to a lesser extent TNF-alpha, but not FasL or ROI, to kill E1A-expressing cells. Through the use of S-nitroso-N-acetylpenicillamine, which releases NO upon exposure to an aqueous environment, E1A was shown to directly sensitize tumor cells to NO-induced death. E1A sensitized tumor cells to lysis by macrophages despite up-regulating the expression of 70-kDa heat shock protein. In summary, E1A, but not E7, sensitized tumor cells to lysis by macrophages. Macrophages killed E1A-expressing cells through NO- and TNF-alpha-dependent mechanisms.