Transcript PMM1 - Ipatimup

Functional diversification after
gene duplication:
the PMM1 and PMM2 case
Rita Quental
Porto, 2010
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Gene duplication
Gene duplication is a major source of genetic and functional diversity
during evolution (Ohno, 1970).
Classical Model
Duplication-degenerationcomplementation model
Duplication
Duplication
Nonfunctionalization
Neofunctionalization
Subfunctionalization
adapted from Force et al, 1999
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The PMM1 and PMM2
genes
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PMM1 and PMM2
Congenital Disorders of Glycosylation (CDG)
Most frequent type: CDG-Ia
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Phosphomannomutase (PMM) deficiency
Man-6-P
PMM
Man-1-P
(van Shaftingen et al, 1995)
GDP-Man
Glycans
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Characterized by the absence of oligosaccharide chainsin glycoconjugates
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Multisystem disorder with a highly variable phenotype
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Neurological involvement is predominant
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20% mortality in the first 5 years
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Disease locus in chromosome region 16p13.3
(Martinsson et al, 1994)
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PMM1 and PMM2
The search for the gene causing CDG-Ia, based on the sequence similarity to
yeast pmm (SEC53), led to the discovery of two genes in the human genome that
encode PMMs.
PMM1
22q13
Not implicated in CDG-Ia
PMM2
16p13
Mutated in CDG-Ia patients
66% aa sequence identity
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PMM1 and PMM2
PMM deficiency (mouse models and human patients)
Pmm2 knockout in mice causes early embryonic lethality
Pmm1-deficient mice are phenotypically normal
(Thiel et al, 2006)
(Cromphout et al, 2006)
In humans, the most frequent PMM2 mutation (R141H) has never been
found in the homozygous state. This mutation leads to a virtually inactive
protein.
PMM1 is not implicated in human disorders
Complete loss of PMM2 activity is incompatible with life
PMM1 is not able to compensate the reduced PMM2 activity
Different pattern of expression?
Functional differences?
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PMM1 and PMM2
Expression analysis
PMM2-
ubiquitously expressed;
lower expression in brain
PMM1-
highly expressed in brain,
but also present in other tissues
Matthijs et al, 1997
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PMM1 and PMM2
Kinetic studies
Step 1
Hexose-1,6-P2 + PMM
Step 2
PMM-P + Man-6-P
Hexose-P + PMM-P
PMM•Man-1,6-P2
PMM-P + Man-1-P
PHOSPHOMANNOMUTASE
PMM2 and PMM1 act as phosphomannomutase essentially with the same rate.
Step 1
Hexose-1,6-P2 + PMM
Step 3
PMM-P + H2O
Hexose-P + PMM-P
PMM + Pi
PHOSPHATASE
PMM1, but not PMM2, has a significant phosphatase activity on the hexose 1,6bisphosphates (representing 3% of the PMM activity)
Pirard et al, 1999
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PMM1 and PMM2
PMM1 phosphomannomutase activity: 28 mol/min/mg of protein
PMM1 phosphatase activity: 2.1 mol/min/mg of protein
IMP (inosinic acid) increases the glucose-1,6-bisphosphatase
activity of PMM1 up to 110-fold, while inhibiting the
phosphomutase activity.
PMM2 has no phosphatase activity and was insensitive to IMP.
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PMM1 and PMM2
Glc-1,6-P2
• ubiquitously present in tissues, but its concentration is particularly
elevated in brain (100 μM)
• cofactor of sugar phosphomutases
• modulates the activity of several enzymes
stimulates- phosphofructokinase and liver pyruvate kinase
inhibits- low Km hexokinases, phosphogluconate dehydrogenase,
and fructose-1,6-bisphosphatase
IMP
• undetectable in brain
• concentration increases in anoxia
Glc-1,6-P2, IMP and PMM1 could have a role in controlling the energy
metabolism of the brain.
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PMM1 and PMM2
PMMs structure
Homodimeric proteins
Haloacid dehalogenase superfamily (HADSP)
• requires a Mg2+ cofactor for catalysis
• four conserved sequence motifs:
• two structural domains: Core - contains the catalytic residues
Cap - substrate specificity loops
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PMM1 and PMM2
The catalytic cycle is accompanied by conformational changes induced by substrate
binding
Positively charged surfaces at the
interface of the cap and core
domains act as an electrostatic
wedge: cap-open conformation.
CAP
CORE
Binding of the negatively charged
substrate would mitigate the
electrostatic repulsion of the cap
and core domains, favoring cap
closure: cap-closed conformation.
Silvaggi et al, 2006
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Question
Which are the molecular mechanisms responsible for the
shift in the substrate binding affinities after the duplication
event?
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Results
Sequences retrieval
Blast search using human PMM1 and PMM2 protein sequences as queries
Total of 46 proteins from 26
animal species
Alignment: MUSCLE software
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Results
Results
Bayesian phylogenetic analysis
Duplication
event
Results
PMM1-fast evolving after duplication
PMM2-slow evolving after duplication
Functional divergence analysis
This approach is based on the assumption that functionally important sites are
under strong purifying selection and therefore evolve slowly.
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Results
Functional divergencerelated
Method developed by Knudsen et al (2001; 2003), that uses Likelihood- Ratio
Tests (LRT) to detect significant rate differences at specific sites in proteins.
•Type I sites:
very conserved in one gene cluster but highly
variable in the other;
•Type II sites:
are very conserved in both genes, but for nonconservative residues;
•Type I/II sites
• Conserved sites
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Results
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PMM1
147
154
183
186
Results
Man1P
PMM2
R183
M186
L147
S154
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Results
Conformal alterations induced by substrate binding
Superimposition of PMM1 unbound (PDB 2FUC; yellow) and bound with Man-1-P
(PDB 2Q4R; grey)
Arg183 and Met186 are Type II functional divergence-related sites
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Results
Molecular docking analysis
+
• Structure of
protein
• Structure of
ligand
• Binding energy
• Binding pocket
Measure the binding energies between different PMM proteins and
substrates (Man-1-P and IMP)
Human PMM1 and PMM2 crystalographic structures are not in the closed
conformation.
Phosphomannomutase from Leishmania mexicana unbound and bound
with Glc-1,6-P2 (Kedzierski et al. 2006)
used as the enzyme-substrate reference model
in the closed conformation
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Results
L. mexicana present either a conservative residue (Val173) or the
same residue (Gln176) as the human PMM2 (Ile174 and Gln177).
Leishmania PMM- green
Human PMM2- grey
Leishmania PMM- green
Human PMM1- yellow
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Results
Binding energy (Kcal/mol) between different PMM proteins and Man-1-P,
IMP and Glc-1,6,-P2
Binding affinity of L. mexicana PMM for IMP is similar to that of Glc-1,6-P2,
while it is lower for Man-1-P.
The bound human PMM1 structure has binding energy values similar to that
of L. mexicana PMM.
Ancestral PMM protein had the ability to accommodate both
substrates.
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Results
The Arginine and Methionine at position
183 and 186, respectively, in the lineage
leading to PMM1 could have improved the
catalytic efficiency towards IMP by
optimizing the active site chemistry and
also
by
stabilizing
the
required
conformation.
Leishmania PMM- green
Human PMM1- yellow
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Conclusions
Conclusions
• PMM1 and PMM2 were originated in a duplication event prior to
vertebrates evolution
• Several positions are potentially related with the functional
diversification of both proteins, and two of them - sites 183 and 186 in
human PMM1 - seem to have played an important role in this process.
• Ancestral PMM protein had most likely the ability to accommodate
both mannose-phosphate and IMP substrates.
• The mutations occurred in the lineage leading to PMM1 in positions
183 and 186 must have enabled an improved sensibility towards IMP.
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Acknowledgments
Prof. António Amorim
Doutora Luísa Azevedo
Ana Moleirinho
Population Genetics Group
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