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Proteomics in Malaria Parasites: Packed with Potential!

Proteomics in Malaria Parasites: Packed with Potential!. Proteomics in Malaria Parasites: Packed with Potential!. Janette Reader University of Pretoria Department of Biochemistry. Janette Reader University of Pretoria. MALARIA The most devastating tropical infectious disease.

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Proteomics in Malaria Parasites: Packed with Potential!

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  1. Proteomics in Malaria Parasites: Packed with Potential! Proteomics in Malaria Parasites: Packed with Potential! Janette Reader University of Pretoria Department of Biochemistry Janette Reader University of Pretoria

  2. MALARIA The most devastating tropical infectious disease • 90% of disease burden in sub-Saharan Africa 300 million people infected annually 1 million deaths 88% children under the age of 5 PROBLEM: Increasing drug and insecticide resistance of the malaria parasite and mosquito vector

  3. BIOLOGICAL CONTROL

  4. BIOLOGICAL MECHANISMS FOR PARASITE CONTROL Genome Transporters 23 megabase ER & Golgi Nucleus Food vacuole Transcriptome 5332 transcripts Mitochondrion Apicoplast Proteome Parasite Vacuole Erythrocyte ~5300 proteins

  5. Transcriptional machinery Regulation of transcription Transcriptional inheritance Transcriptomics Posttranscriptional regulation Posttranslational repression Proteomics Bioinformatics Protein function Relationships Regulatory mechanisms Genome-wide questions Functional genomics tools Biological and mechanistic insights Metabolic pathways Identity determination of hypothetical proteins Cell cycle regulators Sex determinants Chemical validation of drug targets Mode-of-action of inhibitory compounds Improved drug target action Gene expression regulators (transcription and translation) Virulence factors Specialisedorganel function and metabolism Damage compensation Lifecycle development (stage-specific expression) Reproduction genes (strategy-specific expression) Drug resistance mechanisms Mechanism of drug action Reponses to environmental stressors Drug target specification Host-specific adaptation and expression Identification of vaccine targets Virulence determinants Severe disease progression in vivo Drug target identification Interactomics Birkholtz et al, Malaria J 2006

  6. FUNCTIONAL GENOMICS GMEP 1955-1969 Florens et al, 2002 Bozdech et al, 2003 Teng et al, 2009

  7. GENE REGULATION IN P. FALCIPARUM GMEP 1955-1969 • Genome of P. falciparum - 22.8 Mb • Encodes for 5300 genes • 14 chromosomes • 80% A+T nucleotide content • Linear mitochondrial genome 6 kb • Circular apicoplast genome 35 kb • 60% of the predicted open reading frames show no sequence similarity to genes from other organisms • Transcripts are only produced when needed during the life cycle of the parasite in a ‘just-in-time’ fashion Bozdech et al, 2003

  8. PROTEOME OF P. FALCIPARUM • Predicted to have about 5300 proteins • Comprehensive description of only 46% of proteome (~2400 proteins) • Proteins • Have long hydrophobic stretches (insoluble) • Have amino acid repeats (Lysine and Asparagine) • Are comparatively large • Are non-homologous • Highly charged • Proteome is complex: stage-specific expression of proteins associated with biological and metabolic changes • 6% common to all stages Florens et al, 2002

  9. INFORMATION GAINED FROM FUNCTIONAL GENOMICS Smitet al., J Prot Res, 2010 Clark et al., Biochem J, 2008 Van Brummelenet al, JBC, 2010

  10. STRATEGY IN OUR LAB GMEP 1955-1969 Birkholtz et al., 2008

  11. PROTEOMIC PROTOCOLS AVAILIBLE Panpumthong (2006), et al (~200 spots) Makanga (2005), et al Nirmalan (2004), et al Aly (2007), et al (~200 spots) Wu (2006), et al

  12. EXPERIMENTAL LAYOUT • Isolation of proteins • Concentration with 2D-Quant • Determination of proteins with difference in abundance using PD Quest • Identification of proteins • Plasmo2D • Mass Spectrometry

  13. QUANTIFICATION OF PROTEINSWork done by Dr Salome Smit Bradford Lowry Bicinchoninic Acid (BCA) 2D Quant kit

  14. EXPERIMENTAL LAYOUT • Isolation of proteins • Concentration with 2D-Quant • Determination of proteins with difference in abundance using PD Quest • Identification of proteins • Plasmo2D • Mass Spectrometry

  15. OPTIMIZATION OF PROTEOMICS Sensitivity of stains on Plasmodial proteins Work done by Dr Salome Smit MS Silver CCB CCB MS Silver SYPRO Ruby Flamingo Pink SYPRO Ruby Flamingo Pink Smitet al., J Prot Res, 2010

  16. SUMMARY OF STAIN PERFORMANCE ON 1-D AND 2-DE WITH PLASMODIAL PROTEINS Smitet al., J Prot Res, 2010

  17. STAGE SPECIFIC PROTEINS Ring stage Trophozoite stage Smitet al., 2010 Smitet al., J Prot Res, 2010

  18. APPLICATION OF THIS METHOD IN OTHER STUDIES IN P. FALCIPARUM • Co-inhibition of AdoMetDC/ODC in P. falciparum (Tharina Van Brummelen) • Mono-functional inhibition of AdoMetDC in P. falciparum (Salome Smit) • Mono-functional inhibition of ODC in P. falciparum (Katherine Clark) • Herbicide derived compounds tested on P. falciparum (Janette Reader & Jeff Verlinden)

  19. ACKNOWLEDGEMENTS • Prof L Birkholtz • Prof AI Louw • Dr Salome Smit • Malaria team • Past and Present

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