Transcript Document

Epigenetic Processes from a
Molecular Perspective
INBRE Meeting
2/16/10
Outline
• Analytical tools to measure epigenetic
transformations
– HPLC
– MS
– X-Ray crystallography
• Targeting epigenetic processes with
small molecule therapeutics
HPLC
QuickTime™ and a
decompressor
are needed to see this picture.
• High performance (or
high pressure) liquid
chromatography
• Main components:
pump, column,
detector
HPLC Solid Phase Materials
• Can vary column size,
surface area, organic
surface material as
needed
Separation with HPLC
• A solvent polarity gradient is used to elute compounds based
on polarity
• Using a “standard” C18 column, highly polar compounds will
have little affinity for the greasy surface and pass through
quickly relative to nonpolar compounds
Tandem HPLC/MS
• By passing HPLC eluent into a mass
spectrometer, masses can be assigned
to peaks as they elute
• For non-tandem systems, peaks can be
collected separately as they elute and
then inserted into a separate MS
MS Techniques
• “Soft” ionization techniques produce only whole molecular
ions, simplifying data analysis
• “Hard” ionization techniques will fragment whole molecular
ions into smaller molecular ions, giving additional structural
detail
MS/MS Peptide Sequencing
Polypeptide backbones fragment in a predictable manner.
Fragment data can be used to reconstruct order of
attachment within peptide/protein
MS/MS Peptide Sequencing
“Ladder” sequencing used to construct primary structure
MALDI-TOF MS
• Matrix Assisted Laser Desorption Ionization
Time of Flight Mass Spectrometry
A “soft” ionization method
Laser energy is used to
vaporize molecules with
the use of an intermediary
matrix molecule
Useful for large
biomolecules otherwise
difficult to vaporize
MALDI-TOF MS
• Matrix Assisted Laser Desorption Ionization
Time of Flight Mass Spectrometry
Examples of MALDI-TOF Data
X-Ray Diffraction Analysis
Epigenetic Regulatory Reactions
• Histone acetylation/deacetylation
• DNA methylation/demethylation
Histone Acetylation
• Promoted by histone acetyltransferase (HAT)
• Acetylation causes opening of histone
protein arms, making DNA in chromatin more
accessible
• Acetylated histones are a mark of gene
activity
Histone Deacetylation
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Promoted by histone deacetylase (HDAC)
Used to silence gene activity
There are 11 known types of HDAC enzymes
Targeting these HDACs specifically is a major
goal of developing a drug therapy
HDAC Inhibitors
• How are HDAC inhibitors used as potential
cancer treatment tools?
• Inhibiting HDAC will result in an increase of
histone acetylation
• Highly acetylated histone tails will induce
genes that suppress the cancer phenotype
• HDAC inhibitors were observed to block
progression of cancer cells in culture
HDAC Inhibitors
• Histone acetylation is suggested to play a
role in memory formation
• HDAC inhibitors are being explored as
treatments for neurodegeneration and
memory loss
MS to Analyze Histone Methylation
• Parent ion mass can be used to determine
quantity of acetylated sites
(COCH3 mass is +43)
• Ladder sequencing techniques can be
used to determine where the acetylated
lysine residues are located within the
primary structure
X-Ray Diffraction to Study HDACs
• Crystal structure of protein allows active
site to be defined in 3D
• Goal of small molecule inhibitor: block
active site of enzyme so natural substrate
(Lysine side chain) can not bind
• Co-crystallization with a bound inhibitor
give direct insight into mode of action of
therapeutic molecule
HDAC Mechanism
• A zinc metalloenzyme
• Activates the hydrolysis of the amide
functional group using a bound water
molecule
HDAC Protein Structure
Cartoon depiction
Surface depiction
HDAC Active Site Geometry
Binding channel mapping
Active site residues
HDAC Inhibitor Examples
HDAC Inhibitor Examples
DNA Methylation
• Methylation patterns are unique in
different tissues
• Active genes are less methylated than
inactive genes
• Methylated regions silence gene
expression by interacting with proteins
and preventing access to DNA
DNA Methylase
• Enzyme substitutes a methyl (CH3) group to
cysteine units of DNA CG islands
(this can be targeted for inhibition)
• Inhibiting this process would cause global
decrease in methylation level of DNA
• In conjunction with other enzymatic tools,
might DNA methylase inhibition be used as a
tool to re-program cells? Revert to
progenetor or stem cell state?
DNA Demethylation
• DNA believed not to be directly demethylated
but rather methylated cysteine unit is
removed entirely and replaced with
unmethylated cysteine unit
(hence no single enzyme to target for
inhibition to perturb this process)
Conclusion
• Epigenetic modifications can be measured
using molecular analysis tools such as
HPLC, MS and X-ray diffraction
• Epigenetic modifications can be targeted
therapeutically with the use of small
molecule enzyme inhibitors