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Biochemical Society Transactions (2000) Volume 28, part 3
CAMP-SPECIFIC PHOSPHODIESTERASE IV,,
EXPRESSION IN A MAMMALIAN SYSTEM
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Conformational studies of the fibrillogenic ABri peptide
in familial British dementia.
N. Korniotis, A. F. Wilderspin
School of Pharmacy, Department of Pharmaceutical and Biological
Chemistry, 29/39 Brunswick Square. London WClN 1AX.
Type IV Phosphodiesterases (PDEs) known as CAMP-specific PDEs
have the role of hydrolysing the second messenger molecule CAMP
to 5'-AMP. The potential for developing PDE inhibitors for the
therapy of asthma. allergy and other inflammatory diseases will be
helped by structural analysis of PDE IV. PDE IV has been found to
be present predominantly in inflammatory cells. and PDE inhibitors
have already been found to suppress various functions of the
inflammatory cells. To date. there have been limited reports on
systems capable ofproducing milligram quantities of PDE, but some
have enabled biochemical studies important for understanding the
oligomerisation, inhibitor binding and function of the enzyme ( I ) .
For high level expression. the PDE IV,, gene met2'RD1 (2) was
cloned into the Semliki Forest Virus vector pSFV-I. This system is
attractive because it is capable of expressing correctly folded
mammalian proteins which are biologically active. Runoff transcripts
were electroporated into Baby Hamster Kidney cells and expression
resulted in 300 i 100 fold increase in enzyme activity compared to
endogenous enzyme. This activity was found to be sensitive to the
potent PDE IV inhibitor Rolipram. For scale up, the ratio of pSFV1 PDE to helper virus RNA for the production of infectious virus was
2: I . The optimum multiplicity of infection of BHK cells and time
allowed for expression was also investigated.
N K is supported by a BBSRC studentship.
We [hank Professor M.D. Houslay for providing the met"RD1 gene
I Rocque. W.J., erul. (1997) Biochemistry 36, 14250-14261
2 Shakur. Y., er ul (1993) Biochem. J. 292,677-686
32
Prediction of the structure, function and cellular
location of proteins encoded by the GS element, a
'pathogenicity island' in MAP Mycobacterium avium
subsp. paratuberculosis.
Joe. M. Sheridan, Tim Bull, Nazira Sumar, Jun Cheng, Michael
Stellakis and John Hermon-Taylor.
Department of Surgery, Jenner Level I , St. George's Hospital
Medical School, Cranmer Terrace, London, SW I7 ORE, UK.
M.ycohucterium uvium subsp. purutuhercu1o.si.s ( M A P ) is a
member of the Mycobucterium uwium complex (MAC) that
causes the inflammatory bowel disease, JohneS: disease, in
livestock. Significantly, there is strong evidence that MAP is also
the causative agent of a similar disease, Crohn's disease, in
humans. One of three major genetic differences between MAP
and non-pathogenic MAC is the GS element. Based on
bioinformatics and protein modelling we have allocated the
function of the GS proteins gsbA. gsbB to the synthesis of GDPP-L-fucose which gsd uses to transfer fucose. forming an inverted
(a)linkage as the terminal sugar moiety of MAP GPL. The
proteins gsc and a gene, mpu, just downstream of GS, are
proposed to methylate and acetylate this terminal fucose residue.
As GPL is one of three major cell wall constituents of the MAC,
and GPL has an immunomodulatory as well as structural role, the
enzymes involved in its synthesis may provide novel drug targets
against MAP and other MAC members.
Based on molecular models of the GS encoded proteins
and mpu, and predictions of their locations in MAP. the GS
protein, gsd, was found to be a good immunostaining target for
MAP and represents a suitable MAP vaccine component.
0 2000 Biochemical Society
Joe. M. Sheridan, Omar M.A. El-Agnaf*, Giuliano Siligardit,
Brian. M. Austen*
Department of Surgery, and *Neurodegeneration Unit, Department
of Surgery, Jenner Level I , St. George's Hospital Medical School,
Cranmer Terrace, London, SW17 ORE, UK. tDept of Pharmacy,
King's College, 150 Stamford Street, London, SEI XWA, UK.
Familial British dementia (FBD), is an autosomal dominant
neurodegenerative disorder, with biochemical and pathological
similarities to Alzheimer's disease. One notable similarity is the
production of an amyloid peptide (ABri) due to point mutation of a
stop codon in the BRI gene. The mutation extends the wild-type
protein, and following proteolytic cleavage, results in the 34
residue ABri peptide isolated from plaques in FBD brain sections.
Examination of the ABri sequence and the limited literature
suggested that a constraining intramolecular disulphide bond might
exist in ABri. Therefore we synthesized oxidized (cyclic) and
reduced forms (linear) of both the ABri peptide and the analogous
wild type peptide. Fibrils were observed by negative-staining EM
of aged solutions of ABri, and Congo Red stained samples of the
same solution exhibited birefringence. Furthermore, CD spectra of
solutions of the cyclic ABri peptide showed p structure, whereas
the reduced form was random coil. Based on literature of amyloid
proteins, and knowledge of P strand register and turn prediction,
we propose a model for ABri monomers that is consistent with a
two or three stranded P-sheet monomer with a predominantly
hydrophobic face for P-sheet aggregation. Further structural
analysis of ABri peptides will complement the study of causes of
dementia in diseases such as Alzheimer's, that are related to
changes in protein aggregation and folding.
34 CRYSTAL STRUCTURE OF TETRADECAMERIC
MYCOBACTERIUM TUBERCULOSIS CHAPERONIN I0
Michael M. Robertsa, Alun R. Cokerb, Anthony R.M. Coated and
Steve P. woodb
"Department of Medical Microbiology, St. George's Hospital Medical
School, Cranmer Terrace, London SW17 ORE, England and hDivision
of Biochemistry and Molecular Biology, School of Biological
Sciences, University of Southampton, Bassett Crescent East.
Southampton SO16 7PX, England