A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEBUnder basal conditions TFEB, a master regulator of lysosomal biogenesis, is sequestered in the cytosol due to mTORC1-dependent phosphorylation at the lysosomal membrane. Nutrient starvation or lysosomal dysfunction inhibit mTORC1 activity and induce nuclear translocation of TFEB inducing target gene expression.

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We have recently observed promising success in a mouse model at treating the metabolic disorder phenylketonuria (PKU) with phenylalanine ammonia lyase (PAL) from R. toruloides and A. variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability and aggregation. We reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Here we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant. Unlike previously published PAL structures, significant electron density is observed for the two active site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and NHS-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylideneimidazole-5-one (MIO) prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure combined with analysis of its physical properties provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.

Mucopolysaccharidosis type I (MPS I) is caused by an inherited deficiency of alpha-L-iduronidase (IDUA). The result is a progressive, lysosomal storage disease with central nervous system (CNS) as well as systemic involvement. To target gene therapy to the CNS, recombinant adeno-associated virus (AAV) vectors carrying IDUA sequence were administered to MPS I mice via injection into cerebrospinal fluid. In contrast to intravenous administration, this intrathecal administration was effective in generating widespread IDUA activity in the brain, with the cerebellum and olfactory bulbs having highest activities. In general, IDUA levels correlated with vector dose, although this correlation was obscured in cerebellum by particularly high variability. High doses of vector (4 x 10(10) particles) provided IDUA levels approaching or exceeding normal levels in the brain. Histopathology indicated that the number of cells with storage vacuoles was reduced extensively or was eliminated entirely. Elimination of storage material in Purkinje cells was particularly dramatic. A lower vector dose (2 x 10(9) particles) reduced both the number of storage cells and the extent of storage per cell, but the effect was not complete. Some perivascular cells with storage persisted, and this cell type appeared to be more resistant to treatment than neurons or glial cells. We conclude that intrathecal administration of AAV-IDUA delivers vector to brain cells, and that this route of administration is both minimally invasive and effective.

A potential splicing factor is encoded by the opposite strand of the trans-spliced c-myb exon ( ABSTRACT We previously established that the expression of a thymic c-myb mRNA species requires the intermolecular recombination of coding sequences expressed from transcriptional units localized on different chromosomes, in both chicken and human. We now report that a putative splicing factor (PR264), extremely well conserved in chicken and human, is encoded by the opposite strand of the c-myb transspliced exon. The PR264 polypeptide, which contains a typical ribonucleoprotein 80 and an arginine/serine-rich domain, is highly homologous to the DrosophUa splicing regulators tra, tra-2, and su(w) and to the human alternative splicing factor