Proteins that Convert from a
Helix to b Sheet: Implications for Folding and Disease. Pp. 339-347.
Michael Groß
The Use of Circular Dichroism in the Investigation of Protein Structure and Function. Pp. 349-384.
Sharon M.
Kelly and Nicholas C. Price
Aldosterone- and
Progesterone-Membrane-Binding Proteins: New Concepts of Nongenomic Steroid
Action. Pp. 385-401.
K. Haseroth,
M. Christ, E. Falkenstein and M. Wehling
Engineering
Novel Bioactive Mini-Proteins from Small Size Natural and De Novo Designed
Scaffolds. Pp. 403-430.
Loïc Martin
and Claudio Vita
[Back to top] PPARa-Mediated Responses in the Rodent Liver: An Holistic Biochemical View.
Carcinogenesis through the direct action of
genotoxic, DNA damaging chemicals is an established and well-studied paradigm.
As yet there are no short term tests available for non-genotoxic rodent
carcinogens that do not damage DNA but cause liver tumours in long term rodent
bioassays. A key aim is to develop short term in vitro screens for the detection of nongenotoxic carcinogens, and
this requires knowledge of the mode or
mechanism of action of this class of chemicals.
The
largest and most chemically diverse family of non-genotoxic hepatocarcinogens
is the peroxisome proliferators (PPs) such as hypolipidaemic fibrate drugs,
plasticizers used in clingwrap/medical tubing and certain pesticides and
solvents. PPs mediate their
biological responses via activation of the transcription factor PPARa (peroxisome proliferator activated receptor a), a member of the nuclear hormone receptor
superfamily. PPARa activation is
responsible for the pleiotropic effects of PPs in rodent liver such as the
induction of enzymes of the b-oxidation
pathway, hepatocyte DNA synthesis, liver enlargement and tumourigenesis.
Although much is known, we are far from defining the key cell cycle regulating
targets of PPs, due perhaps to past limitations of technology. The technology
of proteomics allows quantitative measurement of the expression levels of
potentially thousands of individual genes at the protein level on exposure to
toxic insult. This is predicted to revolutionise the way many biological
systems are investigated. Here we review the current knowledge of proteins
involved in the response to peroxisome proliferators and describe the impact of
proteomics in this field.
[Back to
top] Proteins that Convert from a Helix to
b Sheet:
Implications for Folding and Disease.
The
sequence of a protein normally determines which amino acid residues will form a helices, and which one b sheets, to an extent that allows secondary
structure prediction to be made with a reasonable reliability. Nevertheless,
non-native helical structures are observed during in vitro folding of several model proteins and may even occur
during protein biosynthesis within the ribosomal exit tunnel. Moreover,
non-native b sheet structures
are common in amyloid fibrils formed by a variety of pathogenic and even
non-pathogenic proteins and peptides. In all of these cases, the formation of a helix precedes the appearance of b sheet, which suggests that conversion from
the simpler, more local helix structure to the often more convoluted sheet
architecture during folding and pathogenic misfolding processes could be a
unifying principle of general importance. A better understanding of this
switching process, and the ability to design molecular systems which can be
induced to switch between these conformations will have a significant impact on
fields ranging from fundamental biochemistry through to applied technology and
medicine.
[Back to
top] The Use of Circular
Dichroism in the Investigation of Protein Structure and Function.
Circular
Dichroism (CD) relies on the differential absorption of left and right
circularly polarised radiation by chromophores which either possess intrinsic
chirality or are placed in chiral environments. Proteins possess a number of
chromophores which can give rise to CD signals. In the far UV region (240-180
nm), which corresponds to peptide bond absorption, the CD spectrum can be
analysed to give the content of regular secondary structural features such as a-helix and
b-sheet. The CD spectrum in the near UV region (320-260
nm) reflects the environments of the
aromatic amino acid side chains and thus gives information about the tertiary
structure of the protein. Other non-protein chromophores such as flavin and
haem moieties can give rise to CD signals which depend on the precise
environment of the chromophore concerned. Because of its relatively modest
resource demands, CD has been used extensively to give useful information about
protein structure, the extent and rate of structural changes and ligand binding.
In the protein design field, CD is used to assess the structure and stability
of the designed protein fragments. Studies of protein folding make extensive
use of CD to examine the folding pathway; the technique has been especially
important in characterising “molten globule” intermediates which may be
involved in the folding process. CD is an extremely useful technique for
assessing the structural integrity of membrane proteins during extraction and
characterisation procedures. The interactions between chromophores can give
rise to characteristic CD signals. This is well illustrated by the case of the
light harvesting complex from photosynthetic bacteria, where the CD spectra can
be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll
molecules. It is therefore evident that CD is a versatile technique in
structural biology, with an increasingly wide range of applications.
[Back to top] Aldosterone- and Progesterone-Membrane-Binding Proteins: New Concepts of Nongenomic Steroid Action.
In the classical theory of steroid action
steroids penetrate into cells and bind to intracellular receptors resulting in
modulation of nuclear transcription and protein synthesis within hours. In
addition, rapid actions of steroids have been identified, which are
incompatible with the classic model of steroid action. Specific binding sites
for aldosterone and progesterone have been reported in membrane preparations of
liver, vascular smooth muscle cells and kidney. These sites are discussed to be
involved in rapid nongenomic steroid actions, such as the rapid activation of
the Na+/H+ exchanger and elevation of [Ca2+]i
in vascular smooth muscle cells by aldosterone. In addition, rapid
progesterone-induced increases of [Ca2+]i
have been reported in spermatozoa. A high affinity progesterone-membrane
binding protein from porcine liver has been identified and cloned. The derived
amino acid sequence showed no significant identity with any functional protein
suggesting a binding site completely different to classic progesterone
receptors. These binding sites are possibly involved in rapidly induced meiotic
maturation of amphibian oocytes and the spermatozoan acrosome reactions as
evidenced by recent studies, where the progesterone induced acrosome reactions
and calcium signaling was blocked by a specific antibody raised against the
membrane binding site for progesterone.
In addition to data on
specific steroid binding and rapid steroid signaling in vitro, results of nongenomic steroid effects in vivo are presented and their
physiological relevance are discussed in the review.
[Back to
top] Engineering Novel Bioactive Mini-Proteins from Small Size Natural and De
Novo Designed Scaffolds.
Mini-proteins,
polypeptides containing less than 100 amino acids, such as (animal toxins,
protease inhibitors, knottins, zinc fingers, etc.) represent successful
structural solutions to the need to express a specific binding activity in
different biological contexts. Artificial mini-proteins have also been designed de novo, representing simplified
versions of natural folds and containing natural or artificial connectivities.
Both systems have been used as structural scaffolds in the engineering of novel
binding activities, according to three main approaches: i) incorporation of functional
protein epitopes into structurally compatible regions of mini-protein
scaffolds; ii) random mutagenesis and functional selection of particular
structural regions of mini-protein scaffolds; iii) minimization of protein
domains by the use of sequence randomization and functional selection, combined
with structural information, in an iterative process. These newly engineered
mini-proteins, with specific and high binding affinities within a small size
and well-defined three-dimensional structure, represent novel tools in biology,
biotechnology and medical sciences. In addition, some of them can also be
directly used in therapy or present high potential to serve as drugs. In all
cases, they represent precious structural intermediates useful to identify frameworks
for peptidomimetic design or directly lead to new small organic structures,
representing novel drug candidates. The engineering of novel functional
mini-proteins has the potential to become a fundamental step towards the
conversion of a protein functional epitope or a flexible peptide lead into a
classical pharmaceutical.