Transcript Slide 1

Strategies for Engineered Negligible Senescence
The decay of extracellular matrix proteins:
an inevitable consequence of old age?
Biology
Matthew Collins
BIOARCH
Archaeology
The decay of extracellular matrix proteins:
an inevitable consequence for archaeology
Biology
Matthew Collins
BIOARCH
Archaeology
Outline
‘Non-disease’ ageing – use in
archaeology
Aspartic acid racemization in the extracellular matrix
Aging: ‘non-disease’ manifestations
blood vessel stiffening
decreasing elasticity of lung fiber
skin slacking
collagen cross-linking
eyesight deterioration
‘Non-disease’ ageing:
Non-disease age related damage of
protein
• a universal phenomena of aging
ancient bones
• stripped of biological control
• extreme phenomena observed
Ageing: the archaeological perspective
Passive damage accumulation
• not programmed
Archaeological materials evidence ageing
• bone / shells
within archaeological closed systems
• rates of damage are chemically predictable
• e.g. the extent of protein damage can be
used to date ancient samples….
Types of damage accumulation
Oxidation
• Not a common problem in most
archaeological settings
Non enzymatic Glycation
• Difficult to assess directly due to presence of
products from soil
Deamidation
• Evidenced as an increase in D-Asp
(racemization)
Chain scission
• Difficult to quantify
Deamidation / racemization in the
extracellular matrix
L-Amino acid
D-Amino acid
Racemization pathways
1. Free amino acid racemization
H3C
H
O
O
H3C
OH
-
O
H
OH
CH3
HO
NH2
NH2
L-Ala
Carbanion intermediate
NH2
D-Ala
Universal mechanism; both in free and peptide-bound amino acids
Widely used in kinetic studies
2. Succinimide formation: only for Asp and Asn (Asx)
O
N
H OH
N
O
L-Asp
O
H
H
N
N
O
O
N
N
N
O
O
L-Suc
D-Suc
H OH
N
O
D-Asp
Elastin: increase in D Asp
6.0
Aorta
percentage D aspartic acid
5.0
Lung
4.0
Ligamentum
Flavum
3.0
Skin
2.0
1.0
Total skin
0.0
0
10
20
30
40
50
60
age (years)
70
80
90
100
Elastin: increase in D Asp
consistent rate
0.5
0.5
Skin
0.4
D/L Asx
D/L Asx
0.4
Ligamentum Flavum
0.3
0.2
0.1
total tissue
0.0
0.3
0.2
0.1
total tissue
0.0
0
10 20 30 40 50 60 70 80 90
age (years)
0
10 20 30 40 50 60 70 80 90
age (years)
‘Collagen’
percentage D aspartic acid
dentine
3.0
bone
2.0
cartilage
1.0
skin
0.0
0
10
20
30
40
50
60
age (years)
70
80
90
100
Protein L-isoaspartyl
methyltransferase
Repair accumulates D-Asp
Repair: D-amino acids
O
HN
OH
NH
O
D- succinimidyl
O
D isoaspartyl
+ H2O
O
N
NH
OH
O
NH
HN
O
O
NH2
D aspartyl
O
NH
NH
O
L asparingyl
-NH3
L- succinimidyl
OH
O
NH
HN
O
N
NH
+ H2O
O
L aspartyl
O
HN
O
HN
NH
OH
OCH3
Duncan McLaren
L isoaspartyl
methyl ester
OH
NH
O
Protein repair
methyltransferase L isoaspartyl
Young et al., JBC 2005
Histone
1-35 PEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRKE
36-68 SYSVYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIASE
77-93 ASRLAHYNKRSTITSRE
H2B
wild type mice
0.10
Protein L-isoaspartyl
methyltransferase
deficient mice
1-35
Asx D/L
0.15
0.05
77-93
36-68
0.0
0
10
20
30
40
50
60
age (years)
70
80
90
100
Long-term rates
Ancient bones
Collagen: Polyproline backbone
Part of an aI chain
succinimide
Results: Gly-Pro-Asn-Gly-Pro
Esteric = - 40.4 kcal/mol
Esteric = - 40.3 kcal/mol
12.2 Å
11.8 Å
Esteric = 3.5 kcal/mol
Esteric = 27.5 kcal/mol
11.8 Å
4.7 Å
Unconstrained
Constrained
Long-term collagen decay
Pattern of collagen loss?
Collagen content trabecular bone (Bailey et al., 1999)
Wt percent collagen in bone
25%
20%
?
15%
10%
male
female
5%
0%
0
10
20
30
40
50
60
age (years)
70
80
90
100
But…
failure strength of bone collagen
failure strength of the collagen
network; (MPa)
35
Wang et al., 2002
30
25
20
15
10
5
0
Young
Mid-aged
Elderly
Mechanical properties: loss vs. tensile
strength
100
Collagen suture
80
80
pH 1.6 37°C
60
60
40
40
20
20
0
0
0
1
2
3
4
time (days)
5
6
7
8
percent of original tensile strength
mass percent of original suture
100
Impact of bone collagen ageing?
fusion
loss of collagen mass
New bone collagen decline in strength
of collagen network
Risk of fracture
0
10
20
30
40
50
Age of samples (yrs)
60
70
80
90
100
Estimating rate of hydrolysis
Aspartic rich proteins
DSD
DGD
from Gotliv et al., 2005 ChemBioChem
His
Asp
Ala
Ile
% of original signal
Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr Gly Pro Val
OCG2
Leu Glu Asp Cys Asp Pro Asn Leu Gla Cys Val Gla Arg Lys
OCG4-30
Leu
Pro
Gla
Pro
Tyr Leu Asp His Trp Leu Gly Ala Hyp Ala Pro Tyr Pro Asp
OCG4
Osteocalcin: Asp14-Pro15
100
bone
10
1
0
200
400
time (days)
600
Tyr Leu Asp His Trp Leu Gly Ala Hyp Ala Pro Tyr Pro Asp Pro Leu Gla Pro Lys Arg Gla Val Cys Gla Leu Asn Pro Asp Cys Asp Glu Leu Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr Gly Pro Val
Temperature
OC
Response
dry
OC
~200 hr
~100 hr
0 hr
wet
15-49
15-49
3-48
3-48
2000 3000 4000 5000 6000
2000 3000 4000 5000 6000
m/z
m/z
McNulty et al., 2002
Cleavage around Asp14
Tyr Leu Asp His Trp Leu Gly Ala Hyp Ala Pro Tyr Pro Asp Pro Leu Gla Pro
Product ion spectra of peptide Arg20-Arg43
from human and Neanderthal
100
Modern Homo
50
*
y13
MH+
y14
y1
y2
0 69.0
y3
y4
y6 y7
650.8
y10
y11
y15
y12
1232.6
y17
b18
1814.4
y23
y22
2396.2
% Intensity
100
TOF
collision
50
0
599.0
1683.4
2767.8
3852.2
m/z
4936.6
2978.0
Nielsen-Marsh et al., 2005, PNAS
% Intensity
y9
Product ion spectra of peptide Arg20-Arg43
from human and Neanderthal
100
Modern Homo
50
*
y13
MH+
y14
y1
y2
y3
y4
069.0
y10
y6 y7
650.8
y11
y15
y12
1232.6
m/z
1814.4
y23
y22
2396.2
2978.0
Shanidar Neanderthal
100
% Intensity
y17
b18
MH+
y9
50
*
y23
y13
y2 y3
069.0
y10
y4
y5
650.8
y7
y12
1232.6
m/z
y15
1814.4
2396.2
2978.0
Nielsen-Marsh et al., 2005, PNAS
% Intensity
y9
Conclusions: passive damage
Elastin
• Surprisingly consistent age dependent increase in D-Asp
Collagen
• Tissue specific increase in D-Asp (e.g. not in skin collagen)
• Surprisingly rapid in bone collagen
• Plateaus due to extreme constraints of triple helix
Bone collagen
• Fibrils more intact with age
• Network more brittle
Decreasing turnover with age?
Exponential decline in the failure strength of the collagen
network?
Exacerbated by
• localized shear / loss of collagen – Oc interaction?
Acknowledgements
BioArch
• Dr Stephen Buckley, Dr Enrico Cappellini, Dr Kirsty
Penkman, Hannah Koon, Rebecca Griffin, Mike
Buckley
Former members
• Dr Oliver Craig, Rome, Dr Jen Hiller, Cardiff, Dr
Christina Nielsen-Marsh, Leipzig, Dr Colin Smith,
Stockholm
Peggy Ostrom MSU
Steffi Ritz-Timme, Remer Dobberstein, Dusseldorf
Funding: NERC (UK), Royal Society (UK), Wellcome
Trust (UK), EU