Transcript Slide 1
Protein Aggregation and Disease
Examples of disorders that are involve protein accumulation in the ER: Congenital Hypothyroidism thyroglobulin accumulates (goiter) Liver damage associated with emphysema anti-trypsin accumulates in liver cells Osteogenesis Imperfecta Hereditary Hypofibrinogemia type I pro-collagen fibrinogen
Conformational Diseases Alzheimer’s Disease: Beta amyloid peptide Prion Diseases : Prion proteins (PrP) Parkinson’s Disease: “Lewy Bodies”
a
-synuclein aggregates to form major component of intracellular Amyotrophic lateral sclerosis : intracellular superoxide dismutase Huntington’s Disease: Huntingtin protein that contain long stretches of polyglutamine
rare vs. inherited
Genetic mutation or environmental factors
Protein
Amyloid protein o
2 structure
a transition Tau Huntingtin Dissociation from microtubes Oligomers a -Synuclein Oligomers
Histopathology Disease
Plaque Neurofibrillar Tangle Nuclear Inclusion Alzheimer Disease Frontotemporal dementia Huntington's Disease Lewy body Parkinson's Disease
Hungtington’s Disease: Involves a Cytosolic Protein
Alzheimer’s Disease
Secretase Cleavage of the Amyloid Precursor C99 Fragment to Produce the Amyloid-
Peptide
Ordered Aggregates of Amyloid-
Cytotoxicity and neuronal cell death Amyloid-
Amyloid-
Amyloid Plaque (observed at autopsy following staining of brain tissue)
Extracellular/Lumen
normal cleavage aberrant C99
Alzheimer’s Disease
Neuronal cell death triggered by aggregates of amyloid-
peptide. Long form (A
) especially aggregation-prone.
In simple forms of AD, A
overproduction occurs.
In sporadic AD, “factors” promote amyloid-
aggregation by… increasing amount of amyloid-
decreasing degradation of amyloid-
decreasing transport of the amyloid-
out of the brain increasing A
:A
ratio altering properties of amyloid-
Possible Routes for Amyloid Formation and Cellular Toxicity Unfolded protein Folding intermediate Properly folded protein factors that could promote amyloid formation at any step: aging, stress, local environment, cell type, toxins (e.g., Al(III), etc.
Vulnerable beta sheet structure aggregation “cross beta” fibrils super-aggregates form and other molecules associate resulting in mature plaques cellular toxicity through any of a variety of possible mechanisms
Protein-Ligand Interactions
Protein-Ligand Interactions: Affinity and Specificity
“Affinity” is a description of the tightness of binding. “High affinity” = tight binding = large K eq = large |-
G o | = (usually) long lifetime for the complex.
Proteins are capable of very high binding specificity.
Protein-Ligand Interactions The Simplest Case: 1:1 Stoichiometry R + L RL R: “receptor” L: “ligand”
Equilibrium Constant:
K association = K a = [RL] [R] .
[L] K dissociation = K d = [R] .
[L] [RL] K a = 1/K d
G o association = -
G o dissociation
K
d
or K
a
?
K d is usually used because of its convenient units: mM, m M, or nM
“Dose/Response” concentration (dose).
Measurable output (response) will vary with L D/R maximum when all R is complexed with L… “saturation”
% of maximal possible functional response of R population is directly proportional to:
fraction of R sites complexed (f R ) = [RL] [R] total = [L] K d + [L] When [L] >> K d , then f R approaches 1… leading to maximum response.
This “binding isotherm” equation can be plotted:
1.0
“saturation” 0.5
f
R 0 0 half maximal saturation free ligand concentration required to achieve f R = 0.5 is equal to K d
[L] (free ligand concentration)
Maximum % change in f R per unit [L] is in the 0-0.5 range
Important implications and considerations…
It is free [L] which is being plotted. However, if [L] free then [L] total is approximately = [L] may be plotted.
total , To approach 100% saturation [L] must be >>K d . When [L] is<< 1-2 K d , f R changes steeply.
This equation is a lot like the M-M equation: f R K d
v/V max K m Dose-response and the [L] required to give 50%-maximum effect: K i , LD 50 , I 50 or K apparent
For a 150 lb. patient and … … a 300 g/mol drug that binds to a single protein target.
K
d 1 nM 1 μM 1 mM Milligrams of Drug to Achieve 5 X K d 0.1
100 100,000
Fitting a Model to Data
If binding is involves 1:1 model, then data should be fit by a hyperbola.
1.0
f
R 0 0 [L] (free ligand concentration)
If model is not appropriate, then the model will not be well fit to the data.
1.0
f
R 0 0 [L] (free ligand concentration)
Typical Ranges for Dissociation Constants Enzyme-Substrate Interactions Receptor-Hormone Interactions Antibody-Antigen Interactions 1
m
M to 5 mM 0.01 nM to 10
m
M 0.01 nM to 10
m
M
G o (kcal/mol) -3 to -8 -7 to -15 -7 to -15 Dissociation constants often reflect the physiological concentrations of the ligands involved. Changes in amino acid sequence in a protein can tune affinity up or down.
Making Binding Data Linear
[RL] f R = [R] total Rearrange to: f R /[L] = 1/K d – f R /K d = [L] K d + [L] Scatchard Equation intercept = 1/K d slope = -1/K d f R /[L] 0 0 f R 1.0
Binding affinity and kinetics R + L RL Association rate = k on . [R] .
[L] Dissociation rate = k off .
[RL] at equilibrium: k on .
[R] .
[L] = k off .
[RL] k off k on [R] . [L] = = K d [RL] k on usually 10 5 -10 8 M -.
sec k off usually scales directly with K d : small K d means low k off half-life of complex = t 1/2 = 0.69/k off Affinity of 1 nM means t 1/2 in the range of 10 sec to 2 hours. “Irreversible” Binding…
Myoglobin and Hemoglobin
Myoglobin, Hemoglobin, and Oxygen:
Paradigms for Simple and Complex Binding
Physiologically important Historically important
However…
“Partial pressure” units are unusual Hemoglobin: unusually complex 5 mM concentration (30% w/v)
Myoglobin Hemoglobin
Myoglobin (Mb)
Myoglobin and Hemoglobin
•
monomeric globular protein
•
1:1 binding stoichiometry
•
main function is to carry oxygen from capillaries to tissues Hemoglobin (Hb)
•
Tetramer: two alpha and two beta subunits
•
alpha and beta subunits are homologous
•
alpha/beta structure similar
•
binds up to 4 molecules of O 2
•
binding is cooperative
•
binding also regulated by heteroallostery
•
function is to transport oxygen from lungs to capillaries, also helps carry CO 2 back to lungs
Both have porphryin cofactor. Porphyrin contains an iron Fe(II) ion: complex called “heme”
Binding of oxygen to myoglobin.
Proximal His (F8) = = = C N O Fe Distal His (E7)
P O2 : partial pressure of oxygen
q
: same as f R P 50 : same as K d
Why Myoglobin Does Not Serve as the Primary Oxygen Delivery Vehicle of Blood P O2 in the lungs is 100 torr.
P O2 in capillaries is ca. 30 torr.
Why Myoglobin Does Not Serve as the Primary Oxygen Delivery Vehicle of Blood P O2 in the lungs is 100 torr.
P O2 in capillaries is ca. 30 torr.
Binding that is more complicated than 1:1 receptor:ligand stoichiometry.
The variables…
How many sites for a given ligand?
Are sites identical?
Are sites cooperative?
Is there a second type of ligand that also binds to the receptor?
Does it compete for the same site(s)?
Is there allostery?
1 ligand, 2 sites, homocooperative
:
R + L RL K 1 K 2 K 1 [L] + 2K 1 K 2 [L] 2 f R = 2 .
(1 + K 1 [L] + K 1 K 2 [L] 2 ) RLL
If K a,2 > K a,1 : “positive cooperativity” Homocooperative 2:1 L:R binding is the simplest route to a sigmoidal binding.
f
R
[L] (free ligand concentration)
Hemoglobin has 4 binding sites, which are positively homocooperative.
The Hill equation: [L] a f R = K d,apparent + [L] a f R log = a (1 – f R )
.
log[L] - logK d,apparent
What is the physiological significance of cooperativity in hemoglobin?
Other Allosteric Effectors of Hemoglobin
The O 2 Binding/Delivery Properties of Hb Are Further Optimized By:
H +
:
Bohr Effect
(Negative Allostery)
CO 2
Release of CO 2 into capillaries from respiring tissues lowers pH via conversion to HCO 3 to reinfoce Bohr Effect CO 2 + H 2 O HCO 3 + H +
CO 2
also chemically reacts with Hb in capillaries (Negative Allostery)
Bisphosphoglycerate (BPG).
Binding to Hb: Negative Allostery
Additive Allosteric Effects are Used To Achieve Optimal Binding/Delivery The Bohr Effect Applies in lungs.
Applies in Capillaries
Hemoglobin with bound bisphosphoglycerate at
subunit interface.
Structure of Hemoglobin
a T (low affinity) O 2 H + ,CO 2 , BPG a R (high affinity)
The Monod-Wyman-Changeux Model for Cooperativity in Hemoglobin
Effect of oxygen on heme iron
(Major Trigger for Shift in the T to R State Equilibrium) = = = C N O C FG3 FG2 FG1 F7 Fe Proximal His (F8) F6 Plane of heme N
Effect of oxygen on heme iron
= = = C N O C FG3 Plane of heme FG2 FG1 Fe F7 Proximal His (F8) F6 N
The T to R Cooperative Transition in Hemoglobin
Blue = T state (deoxy) Red = R-state (oxy) and in the / a interface
Deoxyhemoglobin (T State) Oxyhemoglobin (R State)
Regulation of Hemoglobin Low affinity for O 2
T
K eq High affinity for O 2
R
K eq : Depends on concentrations of… O 2 : shifts K eq to the right H + , CO 2 , and BPG: shifts K eq to the left
Lungs: High O2, Low CO2 Capillaries: Lower O2, Lower pH, High CO2 BPG: Promotes O2 dissociation in caps much more than it inhibits O2 binding in lungs.
Purposes for Allostery/Cooperativity in Proteins
Control of Loading/Delivery On/Off Switch Throttle Metabolic Feedback
Relationship of Hemoglobin Genetics and Human Disease ψ
: pseudogenes (not transcribed)
ζ,ε
: embryonic Hb chains : fetal Hb subunit (fetal Hb = a
2
2
) Adult Hb: a
2
2
(96%), a
2 δ 2
(3%), a
2
2
(1%) note that there are four alleles for the a chain, but only a pair for
Hemoglobin-Based Disorders
Thalessemias: Are caused by the failure to produce one or more Hb chains in a functional form.
β-Thalessemia major: failure to produce the Hb β chain. Fatal.
β-Thalessemia minor :ca. 50% of the usual amount of the Hb β. Mild.
Hemoglobin Variants: Hundreds Known Most from missense mutations Most are “neutral mutations ” (a.k.a. “neutral polymorphisms”) Sometimes result in disease… such as sickle cell anemia.
Sickle Cell Anemia
First documented “molecular disease”.
An inherited disorder.
Hemoglobin S: Glu6Val mutation in beta subunit.
Hemoglobin S: it is the deoxy form that is prone to polymerize.
A recessive disorder. (Mutations must be homozygous).
Heterozygous case confers resistance to malaria. 30% of sub-Saharan Africans and 8% of African-Americans carry HbS allele… also common in Latin-Americans No cure yet, but there are therapies (e.g., hydroxyurea).
Today’s “Clinical Correlation”…
S. Edelstein, U. of Geneva
Sir David Weatherall
Br. J. Haematology, 2001
“The one regret is that the remarkable advances in human genetics, and in the evolution of molecular medicine in general… have done little to help patients with these distressing diseases.” “The lack of a more definitive approach to the cure of the haemoglobin disorders, despite so much knowledge about their molecular pathology, should not surprise us. The story of the advancement of medical practice over the last century tells us that there is
always
reach the clinic.” a long lag period before developments in the basic sciences
Basis for Protein Affinity for Cognate Ligands
Total Energy Balance Sheet Between Free and Bound States Is What Counts
Energetic contributors to favorable binding: optimized H-bonds optimized shape optimized electrostatic interactions optimized hydrophobic interactions release of waters from protein binding site upon binding
“induced-fit” vs. “lock and key”
The geometry of iron coordination in oxymyoglobin
.
Proximal His
C O
.
..
O=O
.
.
..
.
Distal His
Snug Fit of Ligand Into Binding Pocket Avidin-Biotin Hydrogen Bonding Network
p53: “Guardian of the Genome”
Mutations in the p53 Tumor Suppressor and Cancer P53: key role in controlling cellular proliferation and in mediating protective responses to cell damage and stress p53’s synthesis and degradation are normally tightly regulated over 50% of all cancers involve p53 mutations there are over 20,000 (!) known cancer-related mutations in this protein p53 dysfunction contributes to out-of-control cellular proliferation and tumor growth p53’s function is a transcription factor p53 is a tetrameric protein
Esophagus Ovary Colorectum Pancreas Head&Neck Lung Skin Bladder Stomach Brain Liver Breast Uterus Soft tissue Prostate Hematol.
Kidney Bone Testis Thyroïde Cervix
TP53 Somatic Mutations are Frequent in Human Cancer
0 5 10 15 20 25 30 40 45 50 Percent of Tumors with a TP53 Mutation
Most mutations are missense (change of 1 amino acid) 80% are mutations in the core (DNA-binding domain). Six sites account for > 20% of cases.
p53 binding to DNA: most common cancer mutation sites
Most Cancer-Linked p53 Mutations Fall Within Five Classes
Specificity of Binding
Protein binding specificity.
Ligand binding specificity.
Achieving Specificity in a Pleiotropic World
Growth Factors (i.e., The Ligands) Different Types of ErbB Family Tyrosine Kinases (Homo- and Heterodimers)