the biomedical relevance of microbial catabolic diversity
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The Biomedical Relevance of Microbial Catabolic Diversity. John Archer Department of Genetics University of Cambridge [email protected]. Free Radical Theory of Aging. Harman, 1956 Auto-oxidative damage ultimately impairs metabolic efficiency - PowerPoint PPT PresentationTRANSCRIPT
The Biomedical Relevance of The Biomedical Relevance of Microbial Catabolic DiversityMicrobial Catabolic Diversity
John ArcherJohn Archer
Department of GeneticsDepartment of Genetics
University of CambridgeUniversity of Cambridge
[email protected]@gen.cam.ac.uk
Free Radical Theory of AgingFree Radical Theory of Aging
Harman, 1956Harman, 1956
Auto-oxidative damage ultimately impairs Auto-oxidative damage ultimately impairs metabolic efficiencymetabolic efficiency
Prediction: promotion of oxidative reactions will Prediction: promotion of oxidative reactions will correlate with reduced longevitycorrelate with reduced longevity
Genetic factors may promote oxidative stressGenetic factors may promote oxidative stress
MetabolismMetabolism
Cells+nutrient+OCells+nutrient+O22-> more cells+CO-> more cells+CO22+H+H22OO
Energy metabolism: derive high energy compounds Energy metabolism: derive high energy compounds from carbon-energy sourcefrom carbon-energy source
Anabolism: complexity of carbon-containing Anabolism: complexity of carbon-containing compounds increasescompounds increases
Catabolism: complexity of carbon-containing Catabolism: complexity of carbon-containing compounds decreasescompounds decreases
Enzyme-catalysed catabolism is highly sensitive to Enzyme-catalysed catabolism is highly sensitive to oxidative modification of substrate because modified oxidative modification of substrate because modified substrates may not bind their cognate enzymesubstrates may not bind their cognate enzyme
Degenerative Molecular Markers: CharacteristicsDegenerative Molecular Markers: Characteristics
Marker often formed by reactive oxygen speciesMarker often formed by reactive oxygen species
Marker concentration should increase with ageMarker concentration should increase with age
Rate of accumulation of the marker should be inversely Rate of accumulation of the marker should be inversely related to longevity of the organismrelated to longevity of the organism
Genetic factors influence rate of accumulationGenetic factors influence rate of accumulation
Aberrant accumulation of marker associated with Aberrant accumulation of marker associated with pathologypathology
Degenerative Molecular Markers: CandidatesDegenerative Molecular Markers: Candidates
LipofuscinLipofuscin
Ceroid-lipofuscinCeroid-lipofuscin
Modified lipids (especially cholesterol) in foam cells Modified lipids (especially cholesterol) in foam cells leading to atherosclerosisleading to atherosclerosis
N-retinyl-N-retinylidene ethanolamine (A2E) in retinal N-retinyl-N-retinylidene ethanolamine (A2E) in retinal pigment epithelial cellspigment epithelial cells
Degenerative Markers or Causative Agent?Degenerative Markers or Causative Agent?
Lipofuscin may not be direct cause of aging. At moderate Lipofuscin may not be direct cause of aging. At moderate levels it has no effect on RER in neurons, but in high levels it has no effect on RER in neurons, but in high levels (75% of pericarion) is deleterious to neuronal levels (75% of pericarion) is deleterious to neuronal adaptability. LSD are strongly linked to ceroid lipofuscin adaptability. LSD are strongly linked to ceroid lipofuscin accumulation.accumulation.
Atheroma is correlated with coronary disease and is a clear Atheroma is correlated with coronary disease and is a clear causative agent.causative agent.
N-retinyl-N-retinylidene ethanolamine (A2E) in retinal N-retinyl-N-retinylidene ethanolamine (A2E) in retinal pigment epithelial cells may have a role age-related pigment epithelial cells may have a role age-related macular degenerationmacular degeneration
Enzyme Addition TherapyEnzyme Addition Therapy
Degenerative marker compounds accumulate because they Degenerative marker compounds accumulate because they are not substrates for normal lysosomal enzymesare not substrates for normal lysosomal enzymes
Degenerative markers do not accumulate in the Degenerative markers do not accumulate in the environment – there must be enzymes which can process environment – there must be enzymes which can process these moleculesthese molecules
Can one identify enzymes from other living systems that Can one identify enzymes from other living systems that can recognise degenerative marker compounds?can recognise degenerative marker compounds?
Brady et al., mannose-terminal glucocerebrosidase Brady et al., mannose-terminal glucocerebrosidase treatment for Gaucher's Disease treatment for Gaucher's Disease
The Substrate LipofuscinThe Substrate Lipofuscin
30-70% protein (standard amino acids)30-70% protein (standard amino acids)
20-50% lipid (triglycerides, fatty acids, cholesterol, 20-50% lipid (triglycerides, fatty acids, cholesterol, phospholipids, dolichol, phosphorylated dolichol)phospholipids, dolichol, phosphorylated dolichol)
Fe, and other heavy metalsFe, and other heavy metals
Autofluorescent compounds 1,4-dihydropyridines, 2-Autofluorescent compounds 1,4-dihydropyridines, 2-hydroxy-1,2-dihydropyrrol-3-ones?hydroxy-1,2-dihydropyrrol-3-ones?
Resistant to lysosomal enzymesResistant to lysosomal enzymes
RhodococcusRhodococcus Metabolic Diversity Metabolic Diversity
Rhodococcus harmless, Gram-positive Rhodococcus harmless, Gram-positive Actinomycete mycolic acid bacteriumActinomycete mycolic acid bacterium
Genome is sequenced >7 MbGenome is sequenced >7 Mb
Thousands of catabolic genes, specific for a vast Thousands of catabolic genes, specific for a vast range of carbon-energy sourcesrange of carbon-energy sources
Aliphatic, halogenated hydrocarbons, halogenated Aliphatic, halogenated hydrocarbons, halogenated aromatics (pentachlorophenol), BTEX, PAH, aromatics (pentachlorophenol), BTEX, PAH, Nitroaromatics, Lignin-related, alkoxy aromatics, Nitroaromatics, Lignin-related, alkoxy aromatics, terephthalates, heteroaromatics, steroids, dioxane, terephthalates, heteroaromatics, steroids, dioxane, tetrahydrofuran etc. etc..tetrahydrofuran etc. etc..
Isolation ProtocolIsolation Protocol
RhodococcusRhodococcus is an oligotrophic bacterium, highly is an oligotrophic bacterium, highly adapted to catabolise complex, recalcitrant mixtures of adapted to catabolise complex, recalcitrant mixtures of substrates simultaneously (no catabolic repression)substrates simultaneously (no catabolic repression)
Provide 80-100 microMolar lipofuscin as sole carbon-Provide 80-100 microMolar lipofuscin as sole carbon-energy source to energy source to Rhodococcus strains. Incubate and Rhodococcus strains. Incubate and score.score.
RhodococcusRhodococcus Catabolism of Lipofuscin Catabolism of Lipofuscin
Demonstrated Demonstrated RhodococcusRhodococcus could utilise lipofuscin, or could utilise lipofuscin, or components of lipofuscin, as a carbon-energy sourcecomponents of lipofuscin, as a carbon-energy source
RhodococcusRhodococcus is a fungal-like bacterium, possesses membrane is a fungal-like bacterium, possesses membrane bound vesicles in which substrates are degraded by membrane bound vesicles in which substrates are degraded by membrane associated enzyme complexesassociated enzyme complexes
It is very probable that the entire spectrum of lipofuscin can be It is very probable that the entire spectrum of lipofuscin can be metabolised by metabolised by RhodococcusRhodococcus
We propose that We propose that Rhodococcus can act as a source of xeno-Rhodococcus can act as a source of xeno-enzymes to augment human metabolismenzymes to augment human metabolism
AtheromaAtheroma
Macrophages enter artery wall to recycle modified Macrophages enter artery wall to recycle modified lipoproteins entrappedlipoproteins entrapped
Recalcitrant modified lipoprotein products accumulate in Recalcitrant modified lipoprotein products accumulate in foam cell lysosomefoam cell lysosome
Lysosomal function impairedLysosomal function impaired
Additional macrophage are recruitedAdditional macrophage are recruited
Aberrant proliferative response by vascular smooth Aberrant proliferative response by vascular smooth muscle cellsmuscle cells
Formation of atherosclerotic plaqueFormation of atherosclerotic plaque
Rhodococcus and AtherosclerosisRhodococcus and Atherosclerosis
Rhodococcus can utilise cholesterol as a sole carbon-Rhodococcus can utilise cholesterol as a sole carbon-energy sourceenergy source
Both extracellular and intracellular membrane bound Both extracellular and intracellular membrane bound cholesterol oxidases are characterisedcholesterol oxidases are characterised
Reaction catalysed by cholesterol oxidase:-Reaction catalysed by cholesterol oxidase:-
Cholesterol ---> 4-cholesten-3-oneCholesterol ---> 4-cholesten-3-one
We propose that Rhodococcus can act as a source of xeno-We propose that Rhodococcus can act as a source of xeno-enzymes to augment catabolism of atherosclerotic plaqueenzymes to augment catabolism of atherosclerotic plaque
Supporting IndicationsSupporting Indications
Cross-talk ProblemsCross-talk ProblemsSubstrate specificity of the bacterial xeno-enzyme will restrict the Substrate specificity of the bacterial xeno-enzyme will restrict the level of cross-talk between the bacterial enzyme and the human level of cross-talk between the bacterial enzyme and the human metabolismmetabolism
Delivery to lysosomal compartmentDelivery to lysosomal compartmentMannose-terminal glucocerebrosidase treatment of Gaucher's Mannose-terminal glucocerebrosidase treatment of Gaucher's DiseaseDisease
Lysosomal targeting by glycosylationLysosomal targeting by glycosylation
Acid pH of lysosomal compartmentAcid pH of lysosomal compartmentEnzyme properties can be engineered Enzyme properties can be engineered in vitroin vitro
Immune responseImmune responseSmall sample data, but promising so farSmall sample data, but promising so far
Steps to Biomedical Application of Steps to Biomedical Application of XenohydrolasesXenohydrolases
Isolate competent enzymes using a genomics approachIsolate competent enzymes using a genomics approach
Engineer the recombinant protein for lysosomal targetingEngineer the recombinant protein for lysosomal targeting
PartnerPartner
Competence assay in cell systemCompetence assay in cell system
Murine testsMurine tests
Assay competence in disease modelsAssay competence in disease models
ConclusionsConclusions
Lipofuscin, a degenerative molecular marker and Lipofuscin, a degenerative molecular marker and component of several lysosomal storage diseases can be component of several lysosomal storage diseases can be catabolised completely or partially by enzyme(s) catabolised completely or partially by enzyme(s) encoded by the bacterium encoded by the bacterium RhodococcusRhodococcus
Rhodococcus can catabolise several components of Rhodococcus can catabolise several components of atheromaatheroma
It is highly likely that recalcitrant lysosomal components It is highly likely that recalcitrant lysosomal components can be removed by xeno-enzyme treatmentcan be removed by xeno-enzyme treatment