7 research outputs found
Gene transfer into the central nervous system using Herpes Simplex Virus-1 vectors
Manipulation of gene expression in developing or in mature central nervous systems (CNS) holds a promise for the resolution of many compelling neurobiological questions, including the feasibility of gene therapy to treat diseases of the brain. In this context, a number of viral vectors has been used in recent years to introduce and express genes into the CNS. This article discusses a gene transfer system based on the Herpes Simplex Virus-1 (HSV-1). We describe here the use of non-replicating, non-toxic HSV-1 vector, 8117/43, in a series of studies carried in our joint program. This vector proves further the utility of HSV-1 as a delivery vehicle to a number of distinct sites within the CNS
Assessment of viral and non-viral gene transfer into adult rat brains using HSV-1, calcium phosphate and PEI-based methods
CNS gene transfer could provide new approaches to the modelling of neurodegenerative
diseases and devising potential therapies. One such disorder is Parkinson’s
disease (PD), in which dysfunction of several different metabolic processes
has been implicated. Here we review the literature on gene transfer systems
based on herpes simplex virus type 1 (HSV-1) and non-viral
polyethyleneimine (PEI) and calcium phosphate nanoparticle methods. We also
assess the usefulness of various CNS gene delivery methods and present some
of our own data to exemplify such usefulness. Our data result from vectors
stereotaxically introduced to the substantia nigra (SN) of adult rats and evaluated
1 week and/or 1 month post injection using histochemical methods to assess
recombinant ß-galactosidase enzyme activity. Gene transfer using PEI or calcium
phosphate-mediated transfections was observed for both methods and PEI was
comparable to that of HSV-1 amplicon. Our data show that the amplicon delivery
was markedly increased when packaged with a helper virus and was similar
to the expression profile achieved with a full-size replication-defective HSV-1
recombinant (8117/43). We also examine whether PEI or HSV-1 amplicon-mediated
gene transfer could facilitate assessment of the biological effects induced
by a dominant negative FGF receptor-1 mutant to model the reduced FGF signalling
thought to occur in Parkinson’s disease
Arginine methylation regulates mitochondrial gene expression in Trypanosoma brucei through multiple effector proteins
Arginine methylation is a post-translational modification that impacts gene expression in both the cytoplasm and nucleus. Here, we demonstrate that arginine methylation also affects mitochondrial gene expression in the protozoan parasite, Trypanosoma brucei. Down-regulation of the major trypanosome type I protein arginine methyltransferase, TbPRMT1, leads to destabilization of specific mitochondrial mRNAs. We provide evidence that some of these effects are mediated by the mitochondrial RNA-binding protein, RBP16, which we previously demonstrated affects both RNA editing and stability. TbPRMT1 catalyzes methylation of RBP16 in vitro. Further, MALDI-TOF-MS analysis of RBP16 isolated from TbPRMT1-depleted cells indicates that, in vivo, TbPRMT1 modifies two of the three known methylated arginine residues in RBP16. Expression of mutated, nonmethylatable RBP16 in T. brucei has a dominant negative effect, leading to destabilization of a subset of those mRNAs affected by TbPRMT1 depletion. Our results suggest that the specificity and multifunctional nature of RBP16 are due, at least in part, to the presence of differentially methylated forms of the protein. However, some effects of TbPRMT1 depletion on mitochondrial gene expression cannot be accounted for by RBP16 action. Thus, these data implicate additional, unknown methylproteins in mitochondrial gene regulation
The Crystal Structure of α‑Dioxygenase Provides Insight into Diversity in the Cyclooxygenase-Peroxidase Superfamily
α-Dioxygenases (α-DOX) oxygenate fatty acids
into 2(<i>R</i>)-hydroperoxides. Despite the low level of
sequence identity,
α-DOX share common catalytic features with cyclooxygenases (COX),
including the use of a tyrosyl radical during catalysis. We determined
the X-ray crystal structure of <i>Arabidopsis thaliana</i> α-DOX to 1.5 Å resolution. The α-DOX structure
is monomeric, predominantly α-helical, and comprised of two
domains. The base domain exhibits a low degree of structural homology
with the membrane-binding domain of COX but lies in a similar position
with respect to the catalytic domain. The catalytic domain shows the
highest degree of similarity with the COX catalytic domain, where
21 of the 22 α-helical elements are conserved. Helices H2, H6,
H8, and H17 form the heme binding cleft and walls of the active site
channel. His-318, Thr-323, and Arg-566 are located near the catalytic
tyrosine, Tyr-386, at the apex of the channel, where they interact
with a chloride ion. Substitutions at these positions coupled with
kinetic analyses confirm previous hypotheses that implicate these
residues as being involved in binding and orienting the carboxylate
group of the fatty acid for optimal catalysis. Unique to α-DOX
is the presence of two extended inserts on the surface of the enzyme
that restrict access to the distal face of the heme, providing an
explanation for the observed reduced peroxidase activity of the enzyme.
The α-DOX structure represents the first member of the α-DOX
subfamily to be structurally characterized within the cyclooxygenase-peroxidase
family of heme-containing proteins
His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase*
Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into
2R-hydroperoxides. PIOX belongs to the fatty acid α-dioxygenase
family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2).
Although these enzymes share common catalytic features, including the use of a
tyrosine radical during catalysis, little is known about other residues
involved in the dioxygenase reaction of PIOX. We generated a model of linoleic
acid (LA) bound to PIOX based on computational sequence alignment and
secondary structure predictions with COX-1 and experimental observations that
governed the placement of carbon-2 of LA below the catalytic Tyr-379.
Examination of the model identified His-311, Arg-558, and Arg-559 as potential
molecular determinants of the dioxygenase reaction. Substitutions at His-311
and Arg-559 resulted in mutant constructs that retained virtually no oxygenase
activity, whereas substitutions of Arg-558 caused only moderate decreases in
activity. Arg-559 mutant constructs exhibited increases of greater than
140-fold in Km, whereas no substantial change in
Km was observed for His-311 or Arg-558 mutant constructs.
Thermal shift assays used to measure ligand binding affinity show that the
binding of LA is significantly reduced in a Y379F/R559A mutant construct
compared with that observed for Y379F/R558A construct. Although Oryza
sativa PIOX exhibited oxygenase activity against a variety of
14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing
modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these
data suggest that Arg-559 is required for high affinity binding of substrates
to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below
Tyr-379 for catalysis