ASP 53, a 53 kDa cupin-containing protein from Acacia erioloba seeds that protects proteins against thermal denaturation

Includes bibliographical references (leaves 103-111)

Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum</it>, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting <it>P. falciparum </it>spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level.</p> <p>Results</p> <p>Morphological analysis of <it>P. falciparum </it>3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels.</p> <p>Conclusions</p> <p>This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in <it>P. falciparum</it>, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.</p

Isolation and characterisation of a LEA-like protein from yeast (Saccharomyces cerevisiae)

Bibliography: leaves 48-53.LEA proteins are plant proteins that are characteristically hydrophilic and soluble at elevated temperature. The consistent correlation between desiccation tolerance in orthodox seed tissue and an accumulation of LEA proteins suggests that these proteins play an important role in protecting cells from desiccation induced damage. Yeast (Saccharomyces cerevisiae) has been known to desiccate as part of its normal growth cycle and to remain viable after long periods in the desiccated state. As a result of these properties this project was designed to investigate the presence of LEA-like proteins in yeast. A protein was isolated from baker’s yeast that fulfils the requirements for being a LEA protein. This protein, with a molecular mass of 11 kDa, was found to be the most prevalent heat soluble protein in the yeast extract. Antibodies raised against LEA group I proteins recognised this 11 kDa yeast protein in the total extract but failed to recognise the protein after heat treatment at 80°C for 10 min. Amino acid analysis showed that the ll kDa protein was highly hydrophilic - a characteristic of LEA proteins. The protein was partially sequenced (10 cycles) after CNBr digestion and the sequence obtained was compared with the sequence of known proteins in the Stanford databank. Only one protein, HSP 12, was identified to be 100 % homologous to the obtained sequence without the introduction of gaps. Despite a previous report that HSP 12 is a heat shock protein, HSP 12 was present in a reduced concentration in yeast grown at 37 °C compared with yeast grown at 30 °C. HSP 12 was found to increase in concentration after entry into stationary phase - a time when nutrients are limiting and the yeast is preparing to reduce its water content and sporulate. This might be considered equivalent to plant seed maturation - the stage when LEA proteins are synthesised. Moreover, growth conditions that have been reported to stimulate LEA protein biosynthesis in plants also stimulated HSP 12 synthesis in yeast. Purified HSP 12 was shown to inhibit thermal denaturation of yeast alcohol dehydrogenase (ADH) at elevated temperatures. This is a functional property of the pea seed p11 LEA group I protein. From the above results, it was therefore concluded that HSP 12 should be identified as a LEA-like protein rather than as a heat shock protein

ASP53, a thermostable protein from Acacia erioloba seeds that protects target proteins against thermal denaturation.

ASP53, a 53 kDa heat soluble protein, was identified as the most abundant protein in the mature seeds of Acacia erioloba E.Mey. Immunocytochemistry showed that ASP53 was present in the vacuoles and cell walls of the axes and cotyledons of mature seeds and disappeared coincident with loss of desiccation tolerance. The sequence of the ASP53 transcript was determined and found to be homologous to the double cupin domain-containing vicilin class of seed storage proteins. Mature seeds survived heating to 60◦C and this may be facilitated by the presence of ASP53. Circular dichroism spectroscopy demonstrated that the protein displayed defined secondary structure, which was maintained even at high temperature. ASP53 was found to inhibit all three stages of protein thermal denaturation. ASP53 decreased the rate of loss of alcohol dehydrogenase activity at 55◦C, decreased the rate of temperature-dependent loss of secondary structure of haemoglobin and completely inhibited the temperature-dependent aggregation of egg white protein