Showing papers in "Essays in Biochemistry in 1995"

The chloroplast genome.

Abstract: The chloroplast genome consists of homogeneous circular DNA molecules. To date, the entire nucleotide sequences (120-190 kbp) of chloroplast genomes have been determined from eight plant species. The chloroplast genomes of land plants and green algae contain about 110 different genes, which can be classified into two main groups: genes involved in gene expression and those related to photosynthesis. The red alga Porphyra chloroplast genome has 70 additional genes, one-third of which are related to biosynthesis of amino acids and other low molecular mass compounds. Chloroplast genes contain at least three structurally distinct promoters and transcribe two or more classes of RNA polymerase. Two chloroplast genes, rps12 of land plants and psaA of Chlamydomonas, are divided into two to three pieces and scattered over the genome. Each portion is transcribed separately, and two to three separate transcripts are joined together to yield a functional mRNA by trans-splicing. RNA editing (C to U base changes) occurs in some of the chloroplast transcripts. Most edited codons are functionally significant, creating start and stop codons and changing codons to retain conserved amino acids.

Lectins--proteins with a sweet tooth: functions in cell recognition.

Abstract: Lectins, non-enzymic proteins that bind mono- and oligosaccharides reversibly and with high specificity, occur widely in nature. They come in a variety of sizes and shapes, but can be grouped in families with similar structural features. The combining sites of lectins are also diverse, although they are similar in the same family. The specificities of lectins are determined by the exact shape of the binding sites and the nature of the amino acid residues to which the carbohydrate is linked. Small changes in the structure of the sites, such as the substitution of only one or two amino acids, may result in marked changes in specificity. The carbohydrate is linked to the protein mainly through hydrogen bonds, with added contributions from van der Waals contacts and hydrophobic interactions. Coordination with metal ions may occasionally play a role too. Microbial surface lectins serve as a means of adhesion to host cells of viruses (e.g. influenza virus), bacteria (e.g. E. coli) and protozoa (e.g. amoeba): a prerequisite for the initiation of infection. Blocking the adhesion by carbohydrates that mimic those to which the lectins bind prevents infection by these organisms. The way is thus open for the development of anti-adhesive therapy against microbial diseases. Lectin-carbohydrate mediated interactions between leucocytes and endothelial cells are the first step in the recirculation of lymphocytes and in the migration of neutrophils to sites of inflammation. Such interactions may also feature highly in the formation of metastases. Studies of these processes are expected to lead to the development of carbohydrate-based anti-adhesion drugs for the treatment of inflammatory diseases as well as cancer.

Tyrosine hydroxylase: human isoforms, structure and regulation in physiology and pathology.

Abstract: TH is a tetrahydrobiopterin-requiring, iron-containing monooxygenase. It catalyses the conversion of L-tyrosine to L-dopa, which is the first, rate-limiting step in the biosynthesis of catecholamines (dopamine, noradrenaline and adrenaline), the central and sympathetic neurotransmitters and adrenomedullary hormones. The cofactor of TH is tetrahydrobiopterin, which is synthesized from GTP in three steps. The TH gene consists of 14 exons only in humans and 13 exons in animals. Human TH exists in four isoforms (hTH1-4) that are produced by alternative mRNA splicing from a single gene. A single mRNA and protein corresponding to hTH1 exists in non-primates. Monkey TH exists in two isoforms, corresponding to hTH1 and hTH2. TH activity is regulated in the short term by feedback inhibition of catecholamines in competition with tetrahydrobiopterin, and by activation and deactivation due to phosphorylation and dephosphorylation, mainly at Ser-19 and Ser-40 of hTH1. The multiple TH isoforms in humans and monkeys have additional phosphorylation, resulting in more subtle regulation. In long-term regulation under stress conditions, TH protein is induced. CRE and AP1 in the 5' flanking region of the TH gene may be the main functional elements for TH gene expression. TH may be closely related to the pathogenesis of neurological diseases, such as dystonia and Parkinson's disease, psychiatric diseases, such as affective disorders and schizophrenia, as well as cardiovascular diseases. The TH gene may prove useful in gene therapy to compensate for decreased levels of catecholamines in neurological diseases, for example, for supplementation of dopamine in Parkinson's disease.

The roles of molecular chaperones in vivo.

Abstract: Table 1 summarizes the families of chaperones mentioned in this review, and lists their proposed functions. Many of these proteins are named in the accompanying review of Burston and Clarke. Molecular chaperones are proteins which interact with other proteins and help them to reach their final, active conformation. They appear to do this by binding them in an unfolded or partially folded state and subsequently releasing them in an altered form. This property may endow them with several essential or important roles in addition to helping newly synthesized proteins to fold correctly, such as repairing damaged proteins and assisting proteins in membrane translocation. To confirm that a given protein has molecular chaperone activity in vivo, it is necessary to show that interactions between the chaperone and other proteins do occur in the cell, and that loss of the molecular chaperone leads to the accumulation of inactive or precursor protein. The hsp70 protein family are highly conserved and ubiquitous. Genetic studies confirm that their depletion leads to the accumulation of inactive precursor or other proteins, and immunochemical studies show they associate with nascent polypeptides. They are implicated not only in protein folding, but also in protein transport across membranes and reactivation of heat-damaged proteins. The hsp60 proteins are also ubiquitous and very similar in sequence. Those found in bacteria and organelles, such as mitochondria (the GroEL family), are essential at all temperatures, and particularly after heat shock. Their loss or depletion leads to the formation of protein aggregates and eventual cell death. A co-chaperone protein (GroES) is required for their function. Cytosolic homologues (the TCP1 family) are also essential, though not heat-shock induced; they are believed to have a chaperone role in tubulin assembly and their actual role in the cell may be much broader. Many other proteins may have a chaperone function in vivo. Such a function may be specific to a particular substrate (such as the PapD protein in E. coli); others may be more general (such as hsp90 and SecB). Evidence is still needed to demonstrate whether all those proteins which show chaperone behaviour in vitro actually have such a role in vivo. It seems likely that different classes of chaperone may overlap in their specificity, and it is certain that the various proteins classed as molecular chaperones fulfil a wide variety of roles in the cell.

Molecular chaperones: physical and mechanistic properties.

Abstract: Molecular chaperones can be broadly defined as proteins which interact with non-native states of other protein molecules. This activity is important in the folding of newly synthesized polypeptides and the assembly of multisubunit structures; the maintenance of proteins in unfolded states suitable for translocation across membranes; and the stabilization of inactive forms of proteins which are turned on by cellular signals; and the stabilization of proteins unfolded during cellular stress. The major chaperone classes are hsp60 (including TCP1), hsp70 and hsp90. All these proteins prevent the aggregation of unfolded proteins and the strength of interaction with their protein substrates is modified by the binding and hydrolysis of ATP. Hsp70 is a dimeric and ubiquitous protein which binds its substrates in an extended conformation through hydrophobic interactions. It binds to newly synthesized proteins and is required for protein transport. In its ATP-bound state it has a low protein affinity but when the nucleotide is hydrolysed to give the ADP state the affinity is increased. Hsp70 in E. coli (DnaK) is regulated by two co-proteins: DnaJ (of which there are homologues in eukaryotes) stimulates hydrolysis of ATP and GrpE promotes the dissociation of ADP to allow rebinding of ATP. Thus DnaJ promotes the association of substrate proteins and GrpE promotes dissociation. Hsp60 is a large, tetradecameric protein with a central cavity in which non-native protein structures are proposed to bind. It is essential for the folding of a huge spectrum of unrelated proteins and is present in all biological compartments except the ER. As in hsp70, the binding of ATP stimulates release of the substrate and its hydrolysis restores high binding affinity. It functions in conjunction with a co-protein, cpn10, which enhances its ability to eject proteins during the ATPase cycle. The enhancement of folding yields arises either from the prevention of irreversible aggregation or the ability to unfold misfolded structures and allow further attempts to arrive at the native state. Proteins of the hsp90 class are found associated with inactive or unstable substrate proteins within the cell, thus preventing their aggregation and/or permitting rapid activation.

Protein stability at high temperatures

Abstract: The enzymology of hyperthermophilic micro-organisms is a growing field. As increasing numbers of novel high-temperature organisms are isolated and made available through culture collections, and, as biomass becomes more readily available, more laboratories will undoubtedly expand their research interests into this area. The prospect of totally novel enzyme systems and of new approaches to the investigation of fundamental molecular properties will continue to stimulate interest in this field. Studies of thermostable enzymes have already provided valuable data on the relationships between protein stability and activity. The subtle molecular mechanisms which have evolved to stabilize these proteins provide the clues needed for the intelligent design of stabilized mesophilic enzymes, an important target where a combination of high activity at 'low' temperatures and resistance to denaturation is required. The current role of hyperthermophilic enzymes in biotechnology is relatively minor, despite these enzymes having a high 'profile'. While early over-enthusiastic predictions that these enzymes would revolutionize biotechnology should be disregarded, it can reasonably be assumed that, where functional and economic criteria are suitable, thermophilic enzymes will be readily incorporated into current and future biotechnology.

Diphtheria toxin-based receptor-specific chimaeric toxins as targeted therapies.

Abstract: The results from the phase I/II studies of the intravenous administration of DAB486-IL-2 to patients with refractory haematological malignancies have now proven in principle the feasibility of fusion toxin therapy in man. Indeed, the cell-surface receptor-specific intoxication of neoplastic cells through the catalytic ADP-ribosylation of EF-2 is the prototype of a new class of biological response modifiers that may be generally applicable. In those circumstances where either the de novo expression or up-regulation of a cell-surface receptor can be associated with human disease [e.g. the up-regulation of the epidermal growth factor (EGF) receptor on breast cancer], it should be possible to construct genetically a DT-related/growth factor fusion protein to produce an experimental biological treatment of that malignancy. The EGF receptor-targeted fusion toxin DAB389-EGF has within the last year begun human phase I clinical trials. The pre-clinical development of DAB389-IL-7 has begun with the anticipation that this novel fusion toxin will be evaluated in the treatment of the acute leukaemias in which the IL-7R has been shown to be present.