Disentangling the origin of chemical differences using GHOST
Origin and function of a metal resistance gene isolated from eukaryotic soil metatranscriptomes
1. ? ?
ORIGIN(S) & FUNCTION(S) OF METAL RESISTANCE GENES
ISOLATED FROM EUKARYOTIC SOIL METATRANSCRIPTOMES
Supervisor : Laurence Fraissinet-Tachet
Co-supervisor : Roland Marmeisse
ANTOINE ZILLER
3. Soils contained a rich diversity of organisms
This specific biodiversity is organized in complex ecological networks
SOIL BIODIVERSITY
Ecological Networks
4. Soils contained a rich diversity of organisms
This specific biodiversity is organized in complex ecological networks
Eukaryotic organisms play key role in this networks
SOIL BIODIVERSITY
Ecological Networks
Unicellular
eukaryotes
Fungi
5. Chemical pollutions are one of the main anthropic pressure (Rockström et al. 2009)
CHEMICAL POLLUTIONS
6. Chemical pollutions are one of the main anthropic pressure (Rockström et al. 2009)
Oils and metals are the main compounds found on polluted sites
CHEMICAL POLLUTIONS
Polluted sites pollutants
occurrence
61,4% Oils
47,9% Metals
7. Chemical pollutions are one of the main anthropic pressure (Rockström et al. 2009)
Oils and metals are the main compounds found on polluted sites
Metals can be a long term stressor that change ecological networks (Ramade 1992)
CHEMICAL POLLUTIONS
Ecological Networks
Polluted sites pollutants
occurrence
61,4% Oils
47,9% Metals
8. Metals = atoms with a tendency to form cation species (Men+) and be bind to non-metals
METALS IN CELLULAR SYSTEMS
Metals Non Metals
9. Metals Non Metals
Metals = atoms with a tendency to form cation species (Men+) and be bind to non-metals
Reactive metal species (RMS) are involved in every cellular functions
METALS IN CELLULAR SYSTEMS
REACTIVE METAL SPECIES
10. Metals = atoms with a tendency to form cation species (Men+) and be bind to non-metals
Reactive metal species (RMS) are involved in every cellular functions
METALS IN CELLULAR SYSTEMS
REACTIVE METAL SPECIES
Redox chemistry
Energy transfers, cell signaling
Acid-base chemistry
Fe, Cu, Mn, Mo, Ni
Zn2+, Ni2+, Fe3+, Mn3+, Mg2+
Na+, K+, Mg2+, Ca2+, Fe2+, Mn2+, Zn2+
PROTEINS BINDED
MOBILE
Metals Non Metals
11. REACTIVE METAL SPECIES IN CELLULAR SYSTEMS
Reactive metal species in
excess
Nucleic acids, lipids,
proteins, carbohydrates
Cellular system
Metal binding to non-
metal elements
ATOMIC SCALE
12. REACTIVE METAL SPECIES IN CELLULAR SYSTEMS
Reactive metal species in
excess
Nucleic acids, lipids,
proteins, carbohydrates
Cellular system
Enzymatic catalysis
MOLECULAR SCALE
Reactive oxygen
species production
Structural metal
replacement
ATOMIC SCALE
Metal binding to non-
metal elements
13. REACTIVE METAL SPECIES IN CELLULAR SYSTEMS
Reactive metal species in
excess
CELLULAR SCALE
Nucleic acids, lipids,
proteins, carbohydrates
Cellular system
Enzymatic catalysis
MOLECULAR SCALE
Reactive oxygen
species production
Structural metal
replacement
Redox potential and
pH modifications
Metabolic processes
perturbations
ATOMIC SCALE
Metal binding to non-
metal elements
14. REACTIVE METAL SPECIES IN CELLULAR SYSTEMS
Reactive metal species in
excess
CELLULAR SCALE
Nucleic acids, lipids,
proteins, carbohydrates
Cellular system
Enzymatic catalysis
MOLECULAR SCALE
Reactive oxygen
species production
Structural metal
replacement
Redox potential and
pH modifications
Metabolic processes
perturbations
ATOMIC SCALE
Gene induction
PROTEINS INVOLVED IN METAL
RESISTANCE MECHANISMS
Metal binding to non-
metal elements
Metal stress
reduction
17. METAL RESISTANCE MECHANISMS
Cell wall adsorption
Transporters modulation
Organic
compounds
ABC transporters, P1 ATPase
Reactive metal
species
Chitine
18. METAL RESISTANCE MECHANISMS
Cell wall adsorption
Transporters modulation
Intracellular and extracellular binding
Organic
compounds
Metal chelators, Organic acids
ABC transporters, P1 ATPase
Reactive metal
species
Chitine
19. METAL RESISTANCE MECHANISMS
Cell wall adsorption
Transporters modulation
Intracellular and extracellular binding
Organic
compounds
Mercuric reductase
Oxidation state modification
ABC transporters, P1 ATPase
Reactive metal
species
Metal chelators, Organic acids
Chitine
20. METAL RESISTANCE MECHANISMS
Cell wall adsorption
Transporters modulation
Intracellular and extracellular binding
Organic
compounds
Mercuric reductase
Oxidation state modification
ABC transporters, P1 ATPase
Mechanisms mainly described
in vitro with model organisms
In situ
transcriptional responses ?
Reactive metal
species
Metal chelators, Organic acids
Chitine
21. IN SITU TRANSCRIPTIONAL RESPONSE TO METAL POLLUTIONS
EUMETATOX PROJECT
CEFIPRA PROJECT
METAL CONTAMINATED
FORMERLY POLLUTED
NON POLLUTED
Belgian soils
Lehembre et al. 2013 TEAM : EUKARYOTIC MICROORGANISMS,
ADAPTATION TO THEIR ENVIRONMENT
22. IN SITU TRANSCRIPTIONAL RESPONSE TO METAL POLLUTIONS
EUMETATOX PROJECT
CEFIPRA PROJECT
METAL CONTAMINATED
FORMERLY POLLUTED
NON POLLUTED
Belgian soils
Expression in
sensitive yeasts
Vector sequencing from
resistant strains
Lehembre et al. 2013
RNA Extraction
and cDNA libraries
TEAM : EUKARYOTIC MICROORGANISMS,
ADAPTATION TO THEIR ENVIRONMENT
23. TEAM : EUKARYOTIC MICROORGANISMS,
ADAPTATION TO THEIR ENVIRONMENT
IN SITU TRANSCRIPTIONAL RESPONSE TO METAL POLLUTIONS
EUMETATOX PROJECT
CEFIPRA PROJECT
METAL CONTAMINATED
FORMERLY POLLUTED
NON POLLUTED
Belgian soils
RNA Extraction
and cDNA libraries
Expression in
sensitive yeasts
2%
47-63%
35-55%
Vector sequencing from
resistant strains
Sequences comparison with databases
(BLAST)
Lehembre et al. 2013
UNKOWN
GENES
KNOWN GENES
► not as metal
resistance genes
KNOWN GENES
► as metal resistance genes
24. TEAM : EUKARYOTIC MICROORGANISMS,
ADAPTATION TO THEIR ENVIRONMENT
IN SITU TRANSCRIPTIONAL RESPONSE TO METAL POLLUTIONS
EUMETATOX PROJECT
CEFIPRA PROJECT
METAL CONTAMINATED
FORMERLY POLLUTED
NON POLLUTED
Lehembre et al. 2013
Belgian soils
Expression in
sensitive yeasts
2%
47-63%
35-55%
Vector sequencing from
resistant strains
Sequences comparison with databases
(BLAST)
RNA Extraction
and cDNA libraries
ISOLATION OF A CYSTEIN RICH PROTEINS (CRP) FAMILY
UNKOWN
GENES
KNOWN GENES
► not as metal
resistance genes
KNOWN GENES
► as metal resistance genes
41. CRPs vs MTs : CYSTEIN MOTIF OCCURENCE
CRPs have a specific cysteine motif occurrence compared with MTs
CRPs and MTs have the same kind of Cys motifs
45. CRPs vs MTs : CYSTEINE MOTIF ORGANIZATION
CRPs
Bimodular (?) organization and a short linker
CRPs N domain CRPs C domainLinker
46. CRPs vs MTs : CYSTEINE MOTIF ORGANIZATION
CRPs
CCC
CC
CxCC
xxCxx
CxC
xxCxx
Cys motifs are different in N and C domains
CRPs N domain CRPs C domainLinker
Bimodular (?) organization and a short linker
47. CRPs vs MTs : CYSTEINE MOTIF ORGANIZATION
CRPs
CCC
CC
CxCC
xxCxx
CxC
xxCxx
CRPs have specific cysteine motif organization compared with MTs
CRPs N domain CRPs C domainLinker
Bimodular (?) organization and a short linker
Cys motifs are different in N and C domains
48. Expression in E. coli
IN VITRO CHELATION ASSAY
5 cpr genes (4 sub-family)
pGEX-4T-1-CRP (Tag GST)
49. Cd
Zn
Cu
Culture and overexpression
Metal statured medium
Proteins
purification
IN VITRO CHELATION ASSAY
5 cpr genes (4 sub-family)
pGEX-4T-1-CRP (Tag GST)
Expression in E. coli
52. CRPs METAL BINDING ABILITIES
pH 2.4
Intensity
Molecular mass
CRP5-Zn quantification by mass spectrometry
53. CRPs METAL BINDING ABILITIES
pH 2.4
Intensity
Molecular mass
Bind to mixture of Zn species
CRP5-Zn quantification by mass spectrometry
54. CRPs METAL BINDING ABILITIES
pH 2.4
CRP5-Zn quantification by mass spectrometry
Intensity
Molecular mass
Bind to mixture of Zn species
CRP 5 is mainly bind to 9 Zn
60. ARE CRPs METALLOTHIONEINS ?
2 3
CRPs = metallothioneins
1
CRPs = Hypothetical metallothioneins
In vitro chelation of Zn, Cd and Cu
61. ARE CRPs METALLOTHIONEINS ?
2 3
With properties never observed in MTs
large length and low cysteine residues
Cysteine motif occurrence
Bimodular organization with a short linker
CRPs = metallothioneins
1
CRPs = Hypothetical metallothioneins
In vitro chelation of Zn, Cd and Cu
CRPs = new metallothionein familly
62. ARE CRPs METALLOTHIONEINS ?
2 3
With properties never observed in MTs
large length and low cysteine residues
Cysteine motif occurrence
Bimodular organization with a short linker
CRPs = Environmental metallothioneins (EMT)
Ziller et al. 2017
CRPs = metallothioneins
1
CRPs = Hypothetical metallothioneins
CRPs = new metallothionein familly
In vitro chelation of Zn, Cd and Cu
65. Mutant + CRP
Decrease in cell
concentration
Strong
Cd tolerance
– Metal + Metal
CRP 1.1
CRP 1.2
CRP 3
CRP 4
CRP 5
FUNCTIONNAL ORIENTATION
Cd
Zn
Cu
S. cerevisiae
transformation
Tolerant control
*Sensitive control (mutant)
*ABC GSH-Cd vacuolar transporter
*Zn vacuolar transporter
*Cup1 & Crs5 : Metallothioneins
66. Decrease in cell
concentration
Strong
Cd tolerance
– Cd + Cd
CRP 1.1
CRP 1.2
CRP 3
CRP 4
CRP 5
FUNCTIONNAL ORIENTATION
Cd
Zn
Cu
S. cerevisiae
transformation
Tolerant control
*Sensitive control (mutant)
*ABC GSH-Cd vacuolar transporter
*Zn vacuolar transporter
*Cup1 & Crs5 : Metallothioneins
Strong Cd tolerance
Mutant + CRP
67. Mutant + CRP
Decrease in cell
concentration
Strong
Cd tolerance
– Cd + Cd
CRP 1.1
CRP 1.2
CRP 3
CRP 4
CRP 5
FUNCTIONNAL ORIENTATION
Cd
Zn
Cu
S. cerevisiae
transformation
Tolerant control
*Sensitive control (mutant)
*ABC GSH-Cd vacuolar transporter
*Zn vacuolar transporter
*Cup1 & Crs5 : Metallothioneins
Strong Cd tolerance
Weak Zn tolerance
No Cu tolerance
69. FUNCTIONNAL ORIENTATION
Cd
Zn
Cu
S. cerevisiae
transformation
*ABC GSH-Cd vacuolar transporter
*Zn vacuolar transporter
*Cup1 & Crs5 : Metallothioneins
Strong Cd tolerance
Weak Zn tolerance
No Cu tolerance In vitro Cu binding
Ziller et al. 2017
CRPs restore Cd and Zn tolerance
70. Soil with crp sequences
TRANSCRIPTIONAL REGULATION OF CRP GENES
71. Soil with crp sequences
crppromoters
MRE, ARE
?
?
Organism unknown
Promoter region
not available
TRANSCRIPTIONAL REGULATION OF CRP GENES
72. +Cd
In situ gene induction quantification
of soil microcosms
Soil with crp sequences
crppromoters
MRE, ARE
?
?
Organism unknown
Promoter region
not available
TRANSCRIPTIONAL REGULATION OF CRP GENES
73. 3 ppm
6 ppm
12 ppm
24 ppm
CADMIUM TREATMENTS
TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MS
AVAILABLE METALS ESTIMATION
TOTAL METALS
74. 3 ppm
6 ppm
12 ppm
24 ppm
CADMIUM TREATMENTS
TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MSCdmeasured
(ppm)
Cd added
(ppm)
AVAILABLE METALS ESTIMATION
TOTAL METALS
75. 3 ppm
6 ppm
12 ppm
24 ppm
CADMIUM TREATMENTS
AVAILABLE METALS ESTIMATION
TOTAL METALS
Cdmeasured
(ppm)
Cd added
(ppm)
Leaching : 17% Cd initially amended is lost
TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MS
76. 3 ppm
6 ppm
12 ppm
24 ppm
CADMIUM TREATMENTS
Cdmeasured
(ppm)
Cd added
(ppm)
Total Cd is largely available (soil properties)
Leaching : 17% Cd initially amended is lost
TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MS
AVAILABLE METALS ESTIMATION
TOTAL METALS
77. 3 ppm
6 ppm
12 ppm
24 ppm
CADMIUM TREATMENTS
Cdmeasured
(ppm)
Cd added
(ppm)
Natural Cd quantities
0,1-1 ppm
(Alloway et al. 2013)
Biological effects
3-10 ppm
( Smolders 2002)
Total Cd is largely available (soil properties)
Leaching : 17% Cd initially amended is lost
TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MS
AVAILABLE METALS ESTIMATION
TOTAL METALS
78. TRANSCRIPTIONAL REGULATION OF CRP GENES
+Cd
In situ gene induction quantification
of soil microcosms
Soil with crp sequences
crppromoters
MRE, ARE
?
?
Organism unknown
Promoter region
not available
crp sequences are in
low abundance
Soil DNA extract
crp
79. +Cd
Droplet digital PCR ?
In situ gene induction quantification
of soil microcosms
Soil with crp sequences
crppromoters
MRE, ARE
?
?
Organism unknown
Promoter region
not available
crp sequences are in
low abundance
Soil DNA extract
crp
TRANSCRIPTIONAL REGULATION OF CRP GENES
80. Droplet digital PCR is a quantitative PCR based on Poisson statistic, and microfluidic chip
DROPLET DIGITAL PCR
PCR mix partition
in nanoliter droplet
Standard PCR and flow
cytometer Poisson law
81. Droplet digital PCR is a quantitative PCR based on Poisson statistic, and microfluidic chip
ddPCR is more sensitive than qPCR in detecting low quantities of target bacterial DNA in soils
(Kim et al. 2014)
DROPLET DIGITAL PCR
PCR mix partition
in nanoliter droplet
Standard PCR and flow
cytometer Poisson law
82. Droplet digital PCR is a quantitative PCR based on Poisson statistic, and microfluidic chip
ddPCR is more sensitive than qPCR in detecting low quantities of target bacterial DNA in soils
(Kim et al. 2014)
Never use for soil eukaryotic model
DROPLET DIGITAL PCR
PCR mix partition
in nanoliter droplet
Standard PCR and flow
cytometer Poisson law
ddPCR vs qPCR ?
83. +Cd
Droplet digital PCR ?
In situ gene induction quantification
of soil microcosms
Soil with crp sequences
TRANSCRIPTIONAL REGULATION OF CRP GENES
Plasmid crp1 range
101, 102, 103, 104 copy number
84. +Cd
Droplet digital PCR ?
In situ gene induction quantification
of soil microcosms
Soil with crp sequences
TRANSCRIPTIONAL REGULATION OF CRP GENES
Plasmid crp1 range
101, 102, 103, 104 copy number
ddPCR > qPCR
Better efficiency
Lowest measure variability
85. Plasmid crp1 range
101, 102, 103, 104 copy number
+Cd
Droplet digital PCR ?
In situ gene induction quantification
of soil microcosms
Soil with crp sequences
TRANSCRIPTIONAL REGULATION OF CRP GENES
ddPCR > qPCR
Better efficiency
Lowest measure variability
Plasmid crp1 range
101, 102, 103, 104 copy number
+ Microcosm DNA extracts
86. Plasmid crp1 range
101, 102, 103, 104 copy number
+Cd
Droplet digital PCR ?
In situ gene induction quantification
of soil microcosms
Soil with crp sequences
TRANSCRIPTIONAL REGULATION OF CRP GENES
ddPCR > qPCR
Better efficiency
Lowest measure variability
ddPCR and qPCR
amplification perturbations
+ Microcosm DNA extracts
Plasmid crp1 range
101, 102, 103, 104 copy number
87. CRPs FUNCTIONAL ROLE ?
2 31
crp overexpression in Cd, Zn and Cu sensitive yeast
CRPs restore Cd and Zn tolerance
88. CRPs FUNCTIONAL ROLE ?
2 31
crp overexpression in Cd, Zn and Cu sensitive yeast
We have tested crp quantification from microcosms DNA extracts
Need more optimizations to be used in situ
ddPCR is promising method
CRPs restore Cd and Zn tolerance
93. Genome walking
Soil with crp sequences
crppromoters
MRE, ARE
?
?
Organism unknown
Promoter region
not available
Phylogenetic
conserved gene
CRP TAXONOMIC ORIGIN ?
94. Genome walking
Soil with crp sequences
crppromoters
MRE, ARE
?
?
Organism unknown
Promoter region
not available
crp sequences are in
low abundance
Soil DNA extract
crp
Phylogenetic
conserved gene
CRP TAXONOMIC ORIGIN ?
95. Targeted gene captureGenome walking
Soil with crp sequences
crppromoters
MRE, ARE
?
?
Organism unknown
Promoter region
not available
crp sequences are in
low abundance
Soil DNA extract
crp
Phylogenetic
conserved gene
CRP TAXONOMIC ORIGIN ?
96. Sonde ARN
Biotine labeled
probe set
Targeted sequence selective enrichment in environmental DNA extracts (Denonfoux et al.
2013)
TARGETED GENE CAPTURE
crp
Fragmented environmental
DNA extract
97. Sonde ARN
Biotine labeled
probe set
Targeted sequence selective enrichment in environmental DNA extracts (Denonfoux et al.
2013)
TARGETED GENE CAPTURE
crp
DNA-probes
hybridization
Fragmented environmental
DNA extract
98. Sonde ARN
Biotine labeled
probe set
Targeted sequence selective enrichment in environmental DNA extracts (Denonfoux et al.
2013)
TARGETED GENE CAPTURE
crp
DNA-probes
hybridization
Washing and elutionFragmented environmental
DNA extract
Fragmented environmental DNA extract
enriched in targeted sequences
99. Sonde ARN
Biotine labeled
probe set
Fragmented environmental
DNA extract
Targeted sequence selective enrichment in environmental DNA extracts (Denonfoux et al.
2013)
Critical point : probes set design
TARGETED GENE CAPTURE
Fragmented environmental DNA extract
enriched in targeted sequences
crp
DNA-probes
hybridization
Washing and elution
100. CRP SEQUENCES CAPTURE : SET OF PROBES DESIGNED
crp consensus from a 45
sequence alignment
≈ 490 pb
40 probes
50 pb
Specificity
Sensitivity
101. crp consensus from a 45
sequence alignment
≈ 490 pb
40 probes
50 pb
No enrichmentSpecificity
Sensitivity
CRP SEQUENCES CAPTURE : SET OF PROBES DESIGNED
102. crp consensus from a 45
sequence alignment
≈ 490 pb
40 probes
50 pb
No enrichmentSpecificity
Sensitivity
Probes too shorts for long
seize DNA captured ?
CRP SEQUENCES CAPTURE : SET OF PROBES DESIGNED
103. crp consensus from a 45
sequence alignment
≈ 490 pb
40 probes
50 pb
16 probes
327-343 pb
No enrichmentSpecificity
Sensitivity
Specificity
Sensitivity
Probes too shorts for long
seize DNA captured ?
Enrichment x 1 000
CRP SEQUENCES CAPTURE : SET OF PROBES DESIGNED
104. PCR SCREENING AND SEQUENCING RESULTS
Captured
DNA
Cloning and
PCR screening
inserts>1kb
50395
Sequencing
105. PCR SCREENING AND SEQUENCING RESULTS
Captured
DNA
Cloning and
PCR screening
inserts>1kb
50395
Sequencing
33 Crp 4
342
43 Crp 1
333
9 17
crp DNA sequences captured with bording region = success
2 crp sequences
106. PCR SCREENING AND SEQUENCING RESULTS
Captured
DNA
Cloning and
PCR screening
inserts>1kb
50395
Sequencing
crp DNA sequences captured with bording region = success
But they are too short
Gene capture process favor short fragments ?
2 crp sequences
33 Crp 4
342
43 Crp 1
333
9 17
107. CRPs ORIGINE ?
2 31
Crp sequences enrichment of soil DNA extracts
108. CRPs ORIGINE ?
2 31
Cloning/sequencing approach :
2 crp sequences with flanking regions
Crp sequences enrichment of soil DNA extracts
109. CRPs ORIGINE ?
2 31
Genome walking High throughput sequencing
Cloning/sequencing approach :
2 crp sequences with flanking regions
Crp sequences enrichment of soil DNA extracts
111. Origin(s) and function(s) of CRPs ?
GENERAL CONCLUSION
1
Ziller et al. 2017
► Environmental metallothionein (EMT)
CRPs vs MTs
CRPs = new metallothionein family
112. Origin(s) and function(s) of CRPs ?
GENERAL CONCLUSION
2
1
Ziller et al. 2017
Functional characterization
CRPs = role in metal tolerance
Quantitative PCR development (ddPCR) from soil microcosms
► Environmental metallothionein (EMT)
CRPs vs MTs
CRPs = new metallothionein family
113. Origin(s) and function(s) of CRPs ?
GENERAL CONCLUSION
3
2
Taxonomic origin
crp sequence enrichment by gene capture
1
► Environmental metallothionein (EMT)
Ziller et al. 2017
CRPs vs MTs
CRPs = new metallothionein family
Functional characterization
CRPs = role in metal tolerance
Quantitative PCR development (ddPCR) from soil microcosms
118. PERSPECTIVES
CELLULAR SYSTEM
crp organism isolation and
identification
Other crp families
functional role ?
IN SILICO
Isolation of other new CRP in
databases ?
IN VITRO
119. PERSPECTIVES
CELLULAR SYSTEM NATURAL SYSTEM
crp organism isolation and
identification
Other crp families
functional role ?
IN SILICO
Isolation of other new CRP in
databases ?
Importance of organisms
that contain CRP ?
Molecular biosensor tool for
metal pollution detection ?IN VITRO
120. MTs cellular functions are multiple
(Capdevila 2012)
MetallothioneinsMetallothioneins
DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
121. MTs cellular functions are multiple
(Capdevila 2012)
Reactive
metals
ROS
NOS
Metalloprotéines
Autres Molécules
Metallothioneins
Reactive
metals
ROS
NOS
Metalloprotéines
Autres Molécules
Metallothioneins
DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
122. MTs cellular functions are multiple
(Capdevila 2012)
Reactive
metals
ROS
NOS
Metalloprotéines
Autres Molécules
Cell signaling
Cell
detoxification
Redox
equilibrium
Metals
equilibrium
Transcription
regulation
Enzymatic
activity
Metallothioneins
Reactive
metals
ROS
NOS
Metalloprotéines
Autres Molécules
Metallothioneins
DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
123. MTs cellular functions are multiple
(Capdevila 2012)
Reactive
metals
ROS
NOS
Metalloprotéines
Autres Molécules
Cell signaling
Cell
detoxification
Redox
equilibrium
Metals
equilibrium
Transcription
regulation
Enzymatic
activity
Metallothioneins
Reactive
metals
ROS
NOS
Metalloprotéines
Autres Molécules
Metallothioneins
Are MTs a metabolic
hub ?
DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
124. MTs cellular functions are multiple
(Capdevila 2012)
Metabolic hub properties (Cumberworth et al.
2013)
Small linear motifs
Low complexity regions
Reactive
metals
ROS
NOS
Metalloprotéines
Autres Molécules
Cell signaling
Cell
detoxification
Redox
equilibrium
Metals
equilibrium
Transcription
regulation
Enzymatic
activity
Metallothioneins
Reactive
metals
ROS
NOS
Metalloprotéines
Autres Molécules
Metallothioneins
Are MTs a metabolic
hub ?
Cys rich
Cys motifs
DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
126. ? ?
THANK YOU
Supervisor : Laurence Fraissinet-Tachet
Co-supervisor : Roland Marmeisse
ANTOINE ZILLER
Editor's Notes
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
The same set of clusters are found in crustacean and ciliate MT families.We can wonder if the CRPs are just MT sequences from family 3 or 7 which have diverged?
The same set of clusters are found in crustacean and ciliate MT families.We can wonder if the CRPs are just MT sequences from family 3 or 7 which have diverged?
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals.
You always have a control medium without metals.
At the top, positive and negative controls :
-Wild type able to grow
-Sensitive mutant unable to grow
And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals.
You always have a control medium without metals.
At the top, positive and negative controls :
-Wild type able to grow
-Sensitive mutant unable to grow
And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals.
You always have a control medium without metals.
At the top, positive and negative controls :
-Wild type able to grow
-Sensitive mutant unable to grow
And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals.
You always have a control medium without metals.
At the top, positive and negative controls :
-Wild type able to grow
-Sensitive mutant unable to grow
And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals.
You always have a control medium without metals.
At the top, positive and negative controls :
-Wild type able to grow
-Sensitive mutant unable to grow
And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals.
You always have a control medium without metals.
At the top, positive and negative controls :
-Wild type able to grow
-Sensitive mutant unable to grow
And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We analyzed CRP metal binding abilities.
and try to found what are the difference between CRPs and MTs concerning this characteristic ?
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.