Download protein

Nature Reviews Genetics
Yeast
eukaryote model organism
Eukaryote;
– mitochondria,
– organelles,
– cell cycle, etc.
Eukaryote Plus;
– haploid, diploid,
– extra-chromosomal DNA.
Saccharomyces cerevisiae
Baker’s Yeast
Yeast Genome Project
• Yeast Genome Project finished in 1996,
– 1.2 x 107 DNA base pairs,
• 16 chromosomes, 230 kb - 2, 352 kb,
• ~6,000 Open Reading Frames (ORFs),
– Only ~4% of the genes have introns,
• > 70% of the genome is coding.
Yeast Genome Project
vs. human genome
12.1 Mb Genomic DNA sequence (Human, 3,000 Mb)
70% coding sequence (Human, 1.8%)
Few Introns (Humans many)
6012 Genes (Human, 20-25,000)
About 70% of the genes found in humans, are found in yeast.
Known/Unknown
(2001)
3,780 genes with some characterization
560 homologous with other organisms
~1900 unknown
Assigning Gene Function
Geneticist: gene sequence, expression, etc.
Biochemist: enzymatic function,etc.
Cell Biologist: cellular location, etc.
- especially Protein/DNA Interactions
Protein/Protein Interactions
Protein/Membrane Interactions
etc.
The Awesome Power of Yeast Genetics
Homologous Recombination
Transposons
Life Cycle
etc.
Homologous Recombination
• the replacement of a gene with an exogenous gene
through equal crossing over,
homologous region
foreign DNA
homologous region
Before
After
Transposons
Someplace
Transposons: whole units of DNA
that have the ability to insert
themselves into DNA molecules,
– can carry other genes.
Inserts someplace else
Hologous Recombination and Transposons
• Serve as shuttles to carry experimental DNA sequences
into yeast,
– Regulatory sequences (promoters) drive the
expression of,
• Mutant Genes: for structure function analysis,
• Reporter Genes: code for enzymes that signal their presence
in specific cells,
• Epitope Tags: proteins tagged with a foreign peptide sequence
that binds to a specific antibody,
– etc.
Reverse Genetics
Functional Genomics
Gene DNA
Sequence
Gene Disruption
Phenotype
Analysis
Function
Mutate
DNA Sequence
Genetically Link
Development
Physiology
Cell Biology
transp.
lox
…no start codon, no promoter.
transp.
...inserted randomly into a
genomic library.
Haemaglutinin
(HA)
yeast
genomic
library
…+ returns functional transcript, or at least,
an HA tagged peptide that has been targeted.
Fig. 1
?
Biology 470 WEB Page
Up Sides?
Transposon Down Sides
• Insertions are essentially
generated at random;
– it is very difficult to mutagenize
all genes within a genome by
transposon mutagenesis alone,
• but really, transposon-specific
biases in target-site selection,
– for reasons not fully understood,
transposons such as Tn3 and Tn7
insert non-randomly into DNA.
Site Directed Mutagenesis
uptag
downtag
• Systematic deletion of
each ORF in the genome,
– homologous recombination
replaces the gene with a
selectable marker, and a
DNA “barcode”,
• UPTAG,
• DOWNTAG.
Fig. 2
Whole set available:$1,500
Fig. 1. Chemical genomic screening by using a high-density cell array
• “Of the 14 gene deletions that
produce the rapamycin-enhanced
phenotype, 13 genes have human
homologs that showed >30%
identity (highly significant) at the
protein level, and most of them
encode mitochondrial proteins.”
• “Because mitochondrial
dysfunction is known to underlie
the pathogenesis of a wide range of
neurodegenerative disorders…our
result suggests that rapamycin may
be useful in preventing the
progression of these diseases,
including Alzheimer's, Parkinson's,
and Huntington's diseases and
brain aging.”
DNA Microarray
• DNA arrayed at high density
on a solid substrate,
• In this experiment, DNA
complementary to each ORF’s
UPTAG and DOWNTAG is
arrayed in an ordered fashion.
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
Homologous Recombination
UPTAG / DOWNTAG
Fig. 2a
PCR Strategy
Big
Primers
…each strain has one gene KO’d.
Conditional Mutants
…one strain each for >5,900 genes.
Grow deletion strains under
restrictive conditions, PCR U/Dtags, label DNA,
Hybridize/Measure signal,
- absent/altered signal
indicates that the cell with that
particular barcode has low
fitness.
Fig. 2b
Conditional Mutants: mutants that have
observable phenotypes under a given set of
growth conditions.
Formaldehyde cross-link TFs to the DNA...
DNA Protein Interactions
Interactome #1
Epitope tag a transcription
factor of interest.
Shear (cut) genomic DNA
into small fragments.
Fig 3.
cont. next page
DNA Protein Interactions
Interactome #1
Antibodies to the HA
protein are used to collect
the target TF/DNA
fragments.
…target probes from genome with HA-tagged TF,
…reference probes from genome with TF deleted.
SBF, SPO11, etc.
DNA Protein Interactions
Interactome #1
Antibodies to the HA
protein are used to collect
the target TF/DNA
fragments.
The microarray has the
promoters for known genes
arrayed.
Proteomics
Protein-Protein Interactions
Signal Transduction
Pathways,
Yeast Two Hybrid (Y2H),
Heteromeric Protein
Complexes,
Protein Chips (not required),
Allosteric Interactions,
Mass Spectroscopy.
etc.
GAL4 Transcription Activator
native yeast transcription factor
One Protein, Two Functional Domains
BD: Binding Domain,
AD: Activation Domain.
Yeast Life Cycle
Yeast Two Hybrid Vectors
...separate GAL4 Binding Domain and Activation Domain,
...create chimeric proteins, on expression vectors,
– Bait Gene fused to the Binding Domain Gene,
– Target (prey) Gene fused to the Activation Domain Gene.
Yeast Two Hybrid Vectors
…in a diploid cell.
cDNAs are derived from mRNA sequences.
protein of
interest
i.e. constructed
from a cDNA
library.
...mate haploid cells, each expressing the recombinant proteins,
– one with bait,
– the other(s) with prey (target).
No Interaction Bait/Prey
...bait binds DNA,
...prey does not associate with bait, or transcription
machinery.
Bait/Prey Interact
...bait binds DNA,
...prey associates with bait,
...activation domain is then in proximity to transcriptonal machinery,
...reporter gene turned on.
Lot’s of Love; Genetix
• High throughput screening,
• As many as 100,000 matings per day ,
»
• Automatic sample loading, reading and image analysis.
Yeast Interactome
>1,200 Proteins
Two Hybrid Analysis
Single Bait Strategy
What interacts with the protein
implicated in Huntington’s
Disease?
PNAS 100(5):2712-2717
Abstract
• Huntington’s disease (HD) is a neurodegenerative disease
caused by polyglutamine (polyQ) expansion in the protein
huntingtin (htt).
• Pathogenesis in HD seems to involve the formation of
neuronal intranuclear inclusions and the abnormal
regulation of transcription and signal transduction.
• To identify previously uncharacterized htt-interacting
proteins in a simple model system, a yeast two-hybrid
screen was used with a Caenorhabditis elegans “protein
expression” library.
Set-Up
htt
Expressing C.
elegans
proteins.
...mate bait and prey cells, each expressing recombinant proteins,
– diploids that have restored GAL4 activated gene expression contain
peptides that interact.
Bait/Prey Interaction
...found a “C. elegans” protein (K08E3.3b) that interacts
with N-terminal htt in two-hybrid tests.
CIP4 in Human Brains
•
A: Normal, B ---> D increasing
Huntinton’s symptoms.
•
Red Arrows represent CIP4
protein localization. Blue arrow
points to brain lesions.
• A human homolog of the C. elegans K08E3.3b protein is the Cdc42interacting protein 4 (CIP4).
• Neuronal CIP4 immunoreactivity increased with neuropathological
severity in the neostriatum of HD patients.
CIP4 is Sufficient for HD Symptoms
CIP4 protein was over expressed in rat brains.
Cell death and Huntington’s Disease (HD)
morphology resulted.
The Skinny
…and, how many species involved?
• Bait: Human,
• Target (prey): C. elegans (roundworm),
– bait/target match found.
• C. elegans target gene has a human homolog cdc42
interacting protein (CIP4),
– CIP4 found at high levels in HD patient’s brains,
• CIP4 sufficient to cause HD-like symptoms in rats.
Y2H Weaknesses
• False Positives,
– some Baits are “sticky”, sticks to lots of Targets,
– some “Targets” are sticky, sticks to lots of “Baits”,
– fortuitous activation of marker promoter,
• usually assay for multiple markers,
• False Negatives,
– clone fidelity,
– protein conformation (especially membrane bound proteins),
– protein modifications (phosphorylation, glycosylation, etc.),
• Artifacts: Y2H identified interactions require subsequent
confirmation.
Proteomics II
Protein Arrays
Proteomics III
Mass Spectrometry
Proteome
Protein - Protein Interactions
Protein Complexes
Peptide Sequencing
etc.
Mass Spectrometry
• Molecules to be analyzed, referred to as
analytes are first ionized (usually in a
vacuum),
• Newly charged (protonated) molecules are
introduced into an electric and/or
magnetic field in gas phase,
• Their path through the field is a function
of the mass to charge ratio m/z,
• m/z of the ionized species can be used to
deduce the mass of the analyte with high
precision.
Proteomics and Mass Spec
MALDI
ESI-MS
Proteome
Protein - Protein
Protein Complexes
Peptide Sequencing
etc.
Peptide Mass Mapping
“Mass Fingerprinting”
...proteins are cleaved by proteolytic enzymes in a
sequence specific manner,
22.655 kD
8.222 kD
1.457 kD
10.003 kD
13.457 kD
=
One, and only one, 55.792
kD protein in the data base
w/ specific fragment
pattern.
– thus, each protein in a proteome has a unique
peptide mass subset,
• these subsets can be computationally derived
from protein databases, i.e via translated genomic
DNA sequences,
• experimentally determined unknowns can be
compared, via computers, to online databases for
identification,
..scalable, multiple samples can be deposited at
once, computers sort out the constituents.
Tandem Mass Spectroscopy
(MS-MS)
Often provides
enough information
to unambiguously
identify the entire
protein when MS
data is compared to
online databases.
...mass spectrometry can also be used to
obtain sequence to identify peptides,
– treatment with sequence specific protelytic
enzymes provides information on the terminal
residues,
– the mass of the peptide fragment is
determined,
– a short amino acid sequence from the peptide
is obtained.
MS-MS
MS #1: peptide fingerprinting
is performed,
– peptides that have an
appropriate mass for further
study are isolated,
MS #2: selected peptides are
bombarded with argon gas,
making random fractures in
the peptide backbone, and
mass spec is repeated,
- the mass of each of these
fragments is measured.
Mass Difference = Amino Acid Weight
693.37(EYL)1098.55
...single entry in the
database,
+
total peptide mass info
=
TQLYEYLQR
Protein-Protein Interactions
• Interacting proteins are coprecipitated, and excised from
2-D Page gels
– gel slices are run through MSMS,
– computers de-convolute slices
with multiple proteins.
2-D Page
Interaction Mapping
• Multiple proteins
isolated in single gel
slices are candidate
interactors,
• Other experimental
techniques are used to
confirms interactions
(including Y2H).
DNA Damage Repair Network
Yeast Protein
Interactome
Questions
Review
Next
Finish Up, Review
Lectures online at my Course Materials Page.
Read through pp. 579 of the Strategies Paper.