Genetic Buffering

Genetic buffering

5BBG0214/6 Human and Molecular Genetics

Dr. Barry Panaretou

[email protected] 1

How genetic variation in populations is suppressed

Learning outcomes:

•  To understand the concept of suppressing genetic variation by genetic buffering.

•  Identification of the molecular mechanism responsible for genetic buffering.

•  How relaxation of the buffer leads to appearance of phenotypic variation.

•  How genetic buffering affects human health.

2

•  Human genomes contain approximately one single nucleotide polymorphism (SNP) per 1,500 bases

The extent of genetic variation

SNP: chromosomal positions where two or more variant bases exist, each with 1% or > prevalence within a population

•  Two million nucleotide differences per haploid genome o  many genes will have several polymorphic sites

distributed throughout coding and regulatory regions.

•  We do not see an equivalent variation of phenotypes in populations; the effects of much of this genetic variation are suppressed (this is referred to as cryptic genetic variation).

3

Canalization Development is canalized, so that more or less normal organs and tissues are produced despite extensive genetic variation.

Canalized characteristics show reduced variability

4

canalized traits are present in the genetically diverse population of a species

a mechanism must exist to silence the genetic variation and maintain the canalization i.e a genetic buffering mechanism must exist.

This mechanism must have the following characteristics: •  Must play a central role in many key signaling

pathways.

•  The buffer should destabilize under certain conditions to allow appearance of new phenotypes. Ideally should respond to environmental changes to permit evolvability.

•  Should be ubiquitous in nature o  populations of all organisms accumulate genetic

variation because of i) imperfection in the mechanisms that maintain genome fidelity and ii) effect of natural mutagens.

•  In the same genetic background, destabilizing the buffer should always give the same change in morphology (i.e. genetic variation in that background is now exposed).

Identification of the genetic buffering mechanism.

The fruit-fly (Drosophila melanogaster) was used as a model to investigate cryptic variation and how it is suppressed.

Wild populations of flies are remarkably uniform in morphology

..but they are genetically highly diverse…on average there are 6 SNPs per 1,000 base pairs.

..amounting to several hundred thousand SNPs per genome.

Candidates that could operate as a genetic buffer: •  Would have to be a protein(s) that is (are) involved in

many signal transduction and developmental pathways.

•  Activity of the protein must be altered by environmental change, in such a way as to unleash the cryptic variation, leading to new phenotypes.

Molecular chaperones are candidates for the genetic buffering mechanism, because: o  They perform an essential role in cell physiology which

alters significantly during environmental change. o  Fruit-flies with several morphological abnormalities have

already been observed when the function of the Hsp90 chaperone is disturbed.

in vitro: all the information necessary for transformation of a linear polypeptide chain into the final biologically active 3D structure resides in the amino acid sequence

in vivo: protein concentration can reach 300g/L. At any one time too may hydrophobic surfaces will be exposed; most proteins will aggregate rather than fold.

This is prevented by binding of molecular chaperones.

Molecular chaperones: proteins that ensure that the folding of other polypeptides occurs correctly.

The main chaperone families

Most of them originally identified owing to their increased synthesis during heat shock (HSPs: heat shock proteins)

Heat shock proteins of Saccharomyces cerevisiae.

104 90

26

Cytosolic proteins resolved by SDS-PAGE

370C 250C

12

Hsp70: mediates co-translational folding of polypeptides

Hsp70

70

Molecular chaperones: proteins that ensure that the folding of other polypeptides occurs correctly

Hsp (kDa)

Heat shock proteins of Saccharomyces cerevisiae.

104 90

26

Hsp (kDa)

Cytosolic proteins resolved by SDS-PAGE

370C 250C

12

Hsp90

70

Molecular chaperones: proteins that ensure that the folding of other polypeptides occurs correctly

After folding by Hsp70, some proteins are still unstable. Their stability & activity is maintained by subsequent interaction with Hsp90.

In bacteria: HtpG (essential for High TemPerature Growth)

Hsp90 has been conserved throughout evolution: Hsp90 orthologues:

Deletion of both is lethal, so Hsp90 is essential

Hsp90β knock-out in mice is lethal

Hsp90 has been conserved throughout evolution: Hsp90 orthologues:

Grp94 Endoplasmic reticulum Trap-1 Mitochondrial

&

Saccharomyces cerevisiae (bakers yeast): HSC82 (constitutive) HSP82 (induced by stress) In humans: Hsp90β (constitutive) Hsp90α (induced by stress)

In eukaryotes

Hsp90 substrates (‘clients’)

Nuclear hormone receptors: glucocorticoid, oestrogen, androgen but not retinoic acid, thyroid, vitamin D

Other Transcription factors: MyoD, Heat shock transcription factor

Tumour suppressors: p53 (mutant form), Rb

Tyrosine kinases: v-src, fyn, fgr but not Abl

Serine/threonine kinases: Raf, Wee1, Cyclin Dependant Kinases

Others: Nitric oxide synthase, Hypoxia inducible factor 1α, Telomerase

Hsp90 is at the centre of many developmental and signal transduction pathways:

Domains of Hsp90

Hsp90 function is driven by ATP binding and hydrolysis

Geldanamycin competes with ATP for the nucleotide binding pocket (competitive inhibitor).

Why is the Hsp90 mechanism of action subject to intense scrutiny?

1. Most of its identified cellular targets are signal transducers (cell cycle and developmental regulators).

2. It is a drug target.

Mitogen activated protein kinase

cascade RAF

MEK

ERK

RAS

Hsp90

Hsp90

Hsp90

growth factor receptor plasma membrane

ligand

Cell proliferation

nucleus

Signalling cascades are deregulated (permanently

active) in cancer

Hsp90 as a drug target

Numerous components of the cascades are Hsp90

dependent

SRC

Geldanamycin (GA): a potent inhibitor of Hsp90 Originally isolated from Streptomyces hygroscopicus

•  Induces the degradation of proteins mutated in cancer cells such as v-src (preferentially over normal unmutated counterparts)

•  Several major drawbacks as a drug e.g. hepatotoxicity.

•  Because GA Inhibits interaction between Hsp90 and its substrates (e.g. v-src)

Hsp90 as a drug target

RAS

RAF

ERK

MEK

growth factor receptor

Mitogen activated protein kinase

cascade

SRC

plasma membrane

ligand

nucleus cascades no longer operate. Cell

proliferation blocked

GA: inhibitor of Hsp90

Hsp90

Hsp90

Hsp90

Hsp90-dependent proteins no longer fold properly (and then usually degraded).

Why is the Hsp90 mechanism of action subject to intense scrutiny?

1. Most of its identified cellular targets are signal transducers (cell cycle and developmental regulators).

2. It is a drug target.

3. Hsp90 buffers genetic variation…allowing maintenance of phenotypic stability despite extensive genetic variation.

Two observations suggest that Hsp90 buffers genetic variation:

I. The de-stabilizing effect of oncogenic mutations is reversed by Hsp90:

Mutations in RAF (a protein kinase) lead to protein instability which would ordinarily lead to their degradation. The same applies to mutant v-src.

However, the proteins are stable because they interact with Hsp90.

2. Disturbing Hsp90 function in fruit flies leads to morphological abnormalities - affecting virtually every visible structure.

This second observation allowed exploitation of the Drosophila model system to provide more evidence of genetic capacitance by Hsp90.

Approaches undertaken, each approach designed to compromise Hsp90 function:

②  Inhibit Hsp90 by treatment with geldanamycin.

①  Mutate the Hsp90 gene itself: o  fruit flies bore mutations in one of the Hsp90

alleles (mutating both copies is lethal).

deformed fore-leg black facets in eye

notched wings thickened wing veins shortened wings

disorganized abdominal tergites

Examples of the morphological deformities:

Two possible explanations were put forward:

② Compromising Hsp90 may lead to exposure of cryptic genetic variation. Hsp90 may normally suppress expression of genetic variation affecting many developmental pathways.

① Compromising Hsp90 may lead to an increased rate of mutation; Hsp90 may be involved in fidelity of DNA replication.

The more likely of the two is:

② Compromising Hsp90 may lead to expression of cryptic genetic variation. Hsp90 may normally suppress expression of genetic variation affecting many developmental pathways.

I.  The observed phenotypes depend on genetic background of the tested strains. Compromising Hsp90 activity in flies from the same genetic background led to abnormalities in the same tissue or organ.

Whereas an increase in mutation rate would result in a more random distribution of phenotypes.

II.  Compromising Hsp90 function is not mutagenic.

Because:

Is this solely a lab based phenomenon, noticed because the observations were made in flies where Hsp90 was deliberately compromised by mutation or use of an inhibitor?

Can Hsp90 buffer capacity be altered by environmental change without any necessity for mutation of Hsp90?

The genetic background known to give rise to deformed eyes (when Hsp90 is compromised) exhibits the same phenotype when maintained and crossed at elevated temperature (an environmental change known to raise levels of Hsp90).

Yes…

Temperature response curves for deformed eye trait.

Six fly lines used, all of which give rise to deformed eye trait when Hsp90 compromised. Trait appears when temperature is increased (in the absence of Hsp90 mutation). The fly lines which gave rise to the most severe traits when Hsp90 was compromised, also gave the most severe traits when temperature was increased ( , , ).

molecular explanation for environmental changes unleashing genetic variation:

Effect of mutation is buffered at normal temperature.

Less Hsp90 available to stabilize mutant protein A; mutant protein loses function and a phenotype is revealed.

Proteins de-stabilize at high temperature; Hsp90 is required to stabilize them.

Higher temperature:

protein B …stable, low reliance on Hsp90

mutant protein A … mutant, but stabilized by Hsp90

Normal temperature: +ATP/ADP

Compromising Hsp90 in the model plant Arabidopsis thaliana1 in Danio rerio (zebrafish)2 has the same affect

i.e. cryptic genetic variation is revealed

It is likely that Hsp90 is a genetic buffer in all organisms.

1Sangster et al., (2007) PLOS One 7:e648 2Yeyati et al., (2007) PLOS Genetics 3:0431-0447

normal leaf emergence leaf shape shape of cotyledons

In theory, any chaperone could act as a genetic buffer:

In bacteria, GroEL/GroES overexpression doubles the number of mutations in genes encoding substrates of this chaperone1.

1 Tokuriki and Tawfic (2009) Nature 459:669-675.

3. Triose phosphate isomerase (TIM)

ca. 10% of all soluble E. coli proteins are GroEL/GroES dependent

Mutational drifts were induced in:

2. Phosphotriesterase (PTE)

1. GAPDH (glyceraldehydephosphate dehydrogenase) GroEL/GroES

dependent

3. Triose phosphate isomerase (TIM)

2. Phosphotriesterase (PTE)

1. GAPDH (glyceraldehydephosphate dehydrogenase)

o  All Mutants that retained >70% or more activity, were collected in each case.

o  All mutants were transformed into cells which a) overexpressed GroEL/S and b) maintained GroEL/ES at normal levels.

o  These mutants were sequenced.

o  GroES/EL overexpression doubled the number of accumulating mutations in the GroES/EL substrates GAPDH and PTE.

o  In contrast TIM showed a relatively small fraction of variants.

o  The GAPDH mutants (that accumulated when GroES/EL was overexpressed), caused considerable destabilizing effects.

o  This indicates that protein stability is a major constraint in protein evolution, and buffering mechanisms like chaperones alleviate this constraint.

o  A drug that limits genetic buffering would be useful for cancer therapy, as this would destabilize mutated protein and lead to their degradation.

 Consequences:

o Mechanisms that buffer the destabilizing effect of mutations play a role in maintaining higher genetic diversity and enhance the rate of adaptation.

•  Mutant forms of Hsp90 substrates (like RAF and v-src) are orders of magnitude more dependent on Hsp90 than their unmutated counterparts.

•  Differential sensitivity will be exhibited to Hsp90 inhibitors: malignant cells will more susceptible (normal cells will not be affected).

•  Hsp90 inhibitors will destabilize the mutant substrates to a far greater extent (substrates will be degraded).

v-src is undetectable in cancer cells treated with Geldanamycin (i.e. the protein is so unstable it is degraded).

Geldanamycin: a potent inhibitor of Hsp90

…but not useful in the clinic as it is hepatotoxic

•  Retains ability to inhibit Hsp90 & retains potent anti tumour activity..is in clinical trials.

•  Not hepatotoxic

•  17 allyl geldanamycin (17-AG).

R1: CH2-CHCH2NH R2: H R3: OH R4: H

17-AG

R2

R4

R1

R3

…as are 13 other Hsp90 inhibitors:

Trepel et al., (2010) Nature Reviews Cancer 10:537-549

Summary:

•  Cryptic genetic variation exists in genomes.

•  The chaperone Hsp90 can mask the variation. o allowing the build-up and storage of genetic

variation.

•  Environmental change can deplete activity of Hsp90, and the effects of cryptic genetic variation are released.

Rutherford, S.L. and Lindquist, S. (1998) Hsp90 as a capacitor for morphological evolution. Nature 396:336-342

Rutherford, S.L. (2003) Between genotype and phenotype: protein chaperones and evolvability. Nature Reviews Genetics 4:263-274

References: