Current Protein and Peptide Science - Volume 1, Issue 4, 2000

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.

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.

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.

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 (Ca 2+ )i in vascular smooth muscle cells by aldosterone. In addition, rapid progesterone-induced increases of (Ca 2+ )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.

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.