mcb exam iii review ji woong park december 13 th, 2014
TRANSCRIPT
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MCB Exam III Review
Ji Woong ParkDecember 13th, 2014
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Material Coverage
• My review covers 45 points. It’s lectures by Stewart, Huettner, Weihl, Amarasinghe, and Fremont.
• Due to the diverse range of topics (cancer to crystallography), you may need to use slides not in the review to help your understanding.
• But, the exam questions will be based on the review slides. There will be, however, some questions that require solid understanding/application to answer them (hence, the above point)
• Previous years’ exams will be helpful but it’s incorrect to assume they will be like Exam II.
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Terms To Know for “Application” Questions
• RT-qPCR/RNAi/Knockout Gene or Mice• Western Blot/IP/Co-IP• Transfection/Infection• Electrophysiology• Mass Spec/NMR/Crystallography• Site-Directed Mutagenesis• Inhibitors/Dominant Negatives• Fluorescence/Viability/Toxicity Assays
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Telomere Function
distinguishes between the chromosome end and a double strand break
protects the chromosome from end-to-end fusions
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TR
F2
TR
F1
RAP1TPP1
POT1 POT1
TIN2 TRF2
TRF1
TRF2TRF1
An emerging paradigm: the telomere complex does not exclude DNA surveillance, repair and
replication machinery; rather it directs, modulates and specializes the activities of these proteins to
ensure high fidelity replication and telomere stability
Old view DNA repair machinery
New view
DNA repair/replication machinery
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The “telomere” hypothesisP
op
ula
tio
n D
ou
bli
ng
Time
Telo
mer
e L
eng
th
Senescence
1 2 3 4 5 6
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The telomere hypothesisTe
lom
ere
Len
gth
Time
StopRbp53 Senescence
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The telomere hypothesisTe
lom
ere
Len
gth
Time
Continued proliferationRbp53
Crisis
telomere dysfunctiongenetic catastrophecell death
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CAAUCCCAAUC
Telomerase adds telomeric repeats to the 3’ termini of the chromosome
hTERT
hTR
-only the catalytic (hTERT) and RNA (hTR) components are required for activity in vitro
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The telomere hypothesisTe
lom
ere
Len
gth
Time
Senescence
Crisis
Stop Rbp53
1 in ~107
Stable telomere maintenance
hTERT
ALT
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The telomere hypothesisTe
lom
ere
Len
gth
Time
Crisis
Stop
1 in ~107
hTERT
ALT
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ALT
Telo
mer
ase
+
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Telomerase allows ALT cells to form tumors
H-ras
GM847
TUMORS
hTERT
Lacks telomeraseExpresses SV40 Early regionImmortal
Stewart et al 2002
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Extra-telomeric functions of hTERT
Overexpression of mTERT in murine models increases tumor rates in aged mice
Overexpression of hTERT results in resistance to apoptosis
Telomerase is favored over ALT in human tumors
Ectopic expression of mTERT in skin results “hairy” mice, increased stem cell pool
hTERT expression is required in normal fibroblasts to avoid senescence
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ECM Young fibroblastAltered ECM
Senescent fibroblast
Immune cell
Senescent epithelial cell
Cancer cellPreneoplastic cell
Epithelial cell Endothelial cell
Senescence evasion
Tumor
Senescence
Normal
Stromal Promotion
Premalignant
Time/Stress
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For additional reading (highly recommend)
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Types of Stem CellsEmbryonic – from the inner cell mass of pre-
implantation embryos, prior to formation of the 3 germ layers (ectoderm, mesoderm, endoderm)
Somatic – undifferentiated cells found in specific locations in “mature” tissues
iPS cells – induced pluripotent stem cells generated by reprogramming differentiated cells (or cell nuclei, i.e. therapeutic cloning)
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Reprogramming
• SCNT – somatic cell nuclear transfer (reproductive and therapeutic cloning) – deterministic and fairly rapid
• iPS – induced pluripotent stem cells – slow and stochastic (until recently)
• Transdifferentiation – conversion of one terminally differentiated cell type into another without de-differentiation to an immature phenotype. Must rule out cell fusion or other explanations.
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Generating iPS cells
• Express transcription factors: Oct3/4, Sox2, Klf4 and c-Myc (OSKM) OR Oct3/4, Sox2, Nanog and Lin28
• Initial de-differentiation and proliferation (day 1-3, enhanced by Myc); histone modification and chromatin reorganization
• 2nd wave of gene expression - stem cell and development related genes (day 9-12); DNA demethylation and X reactivation
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Transdifferentiation
• Conversion from one differentiated cell type to another without evident de-differentiation and re-differentiation
• Must not be confused by cell fusion or selection for rare pluripotent cells in the source material.
• Induced by expression of transcription factors and microRNAs
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Protein Degradation in the Cell
UPS
Aggresome
Autophagy
Endocytosis
Nucleus
Ub
Ub
Ub
Ub
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Protein Degradation Ubiquitin/Proteasome Pathway
80-90%Most intracellular proteins
• Lysosomal processes10-20%
Extracellular proteinsCell organellesSome intracellular proteins
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UBIQUITIN
K
G
Small peptide that is a “TAG” 76 amino acids C-terminal glycine - isopeptide
bond with the e-amino group of lysine residues on the substrate
Attached as monoubiquitin or polyubiquitin chains
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Ubiquitination of proteins is a FOUR-step process
First, Ubiquitin is activated by forming a link to “enzyme 1” (E1).
Then, ubiquitin is transferred to one of several types of “enzyme 2” (E2).
Then, “enzyme 3” (E3) catalizes the transfer of ubiquitin from E2 to a Lys e-amino group of the “condemned” protein.
Lastly, molecules of Ubiquitin are commonly conjugated to the protein to be degraded by E3s & E4s
AMP
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The proteasomal DUB Usp14 impairs protein degradation
Lee, BH et alNature 467:179-842010
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Autophagy
• Lysosomal degradation of proteins and organelles
• Occurs via three routes– Macroautophagy– Microautophagy (direct uptake of cellular debris
via the lysosome)– Chaperone mediated autophagy (selective import
of substrates via Hsc70 and Lamp2a)
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Selective Autophagy
• Aggregaphagy– p62/SQSTM1, Nbr1• Mitophagy – Parkin, Nix• Reticulophagy – endoplasmic reticulum• Ribophagy – translating ribosomes• Xenophagy – e.g. Salmonella via optineurin• Lipophagy – autophagy mediated lipolysis
• Performed by an expanding group of ubiquitin adaptors
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Rapamycin as an inducer of autophagy
Immunosuppressant used to treat transplant rejection Inhibits the mTOR pathway mTOR integrates extrinsic growth signals and cellular
nutrient status and energy state Active mTOR
Protein synthesis and cell growth Inactive mTOR (or rapamycin treatment)
Inhibition of protein synthesis and increased autophagic degradation of protein
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Protein Structures from an NMR Perspective
Dis
tanc
e fr
om C
orre
ct S
truc
ture
NMR Data Analysis
Correct structure
XNot A Direct Path!
Interpreting NMR Data Requires Making Informed “Guesses” to Move Toward the “Correct” Fold
Initial rapid convergence to approximate correct fold
Iterative “guesses” allow “correct” fold to emerge
Analyzing NMR Data is a Non-Trivial Task!there is an abundance of data that needs to be interpreted
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Protein Structure Determination by NMR
•Stage I—Sequence specific resonance assignment
•State II – Conformational restraints
•Stage III – Calculate and refine structure
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Why use deuteration?
• What are the advantages?
• What are the disadvantages?
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4.1Å
2.9Å
NOE
CaH
NH
NH
CaHJ
NOE- a through space correlation (<5Å)- distance constraint
Coupling Constant (J)
- through bond correlation- dihedral angle constraint
Chemical Shift
- very sensitive to local changes in environment- dihedral angle constraint
Dipolar coupling constants (D)
- bond vector orientation relative to magnetic field- alignment with bicelles or viruses
D
NMR Structure Determination
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Analysis of the Quality of NMR Protein Structures Is the “Average” NMR Structure a Real Structure?
• No-it is a distorted structure level of distortions depends on the similarity between the structures in the
ensemble provides a means to measure the variability in atom positions between an
ensemble of structures Expanded View of an “Average” Structure
Some very long, stretched bonds
Position of atoms are so scrambled the graphics program does not know which atoms to draw bonds between Some regions of the structure
can appear relatively normal
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An 7-step program for protein structure determination by x-ray crystallography
1. Produce monodisperse protein either alone or as relevant complexes
2. Grow and characterize crystals
3. Collect X-ray diffraction data
4. Solve the phase problem either experimentally or computationally
5. Build and refine an atomic model using the electron density map
6. Validation: How do you know if a crystal structure is right?
7. Develop structure-based hypothesis
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1. Produce monodisperse protein either alone or as relevant complexes
Methods to determine protein purity, heterogeneity, and monodispersity Gel electrophoresis (native, isoelectric focusing, and SDS-PAGE) Size exclusion chromatography Dynamic light scattering http://www.protein-solutions.com/
Circular Dichroism Spectroscopy http://www-structure.llnl.gov/cd/cdtutorial.htm
Characterize your protein using a number of biophysical methods
Establish the binding stoichiometry of interacting partners
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2. Grow and characterize crystals
Hanging Drop vapor diffusion Sitting drop, dialysis, or under oil Macro-seeding or micro-seeding Sparse matrix screening methods
Random thinking processes, talisman, and luckThe optimum conditions for crystal nucleation are not
necessarily the optimum for diffraction-quality crystal growth
Space Group P21
4 M3 /ASUdiffraction >2.3Å
14.4% Peg6KNaCacodylate pH 7.0
200mM CaCl2
Space Group P31213 M3 + 3 MCP-1/ASU
diffraction > 2.3Å18% Peg4K
NaAcetate pH 4.1100mM MgCl2
Space Group C22 M3 /ASU
diffraction >2.1Å18% Peg4K
Malic Acid/Imidazole pH 5.1100mM CaCl2
Commercial screening kits available from http://www.hamptonresearch.com; http://www.emeraldbiostructures.com
Hanging Drop Sitting drop
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No Xtals?
Decrease protein heterogeneity
Remove purification tags and other artifacts of protein production
Remove carbohydrate residues or consensus sites (i.e., N-x-S/T)
Determine domain boundaries by limited proteolysis followed by mass spectrometry or amino-terminal sequencing. Make new expression constructs if necessary.
Think about the biochemistry of the system! Does your protein have co-factors, accessory proteins, or interacting partners to prepare as complexes? Is their an inhibitor available? Are kinases or phosphatases available that will allow for the preparation of a homogeneous sample?
Get a better talisman
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3. Collect X-ray diffraction dataInitiate experiments using home-source x-ray generator and detector
Determine liquid nitrogen cryo-protection conditions to reduce crystal decayWhile home x-rays are sufficient for some questions, synchrotron radiation is preferredAnywhere from one to hundreds of crystals and diffraction experiments may be required
Argonne National Laboratory Structural Biology Center beamlineID19
at the Advanced Photon Source http://www.sbc.anl.gov
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4. Solve the phase problem either experimentally or computationally
Structure factor equation:
By Fourier transform we can obtain the electron density.
We know the structure factor amplitudes after successful data collection.
Unfortunately, conventional x-ray diffraction doesn’t allow for direct phase measurement.
This is know as the crystallographic phase problem.
Luckily, there are a few tricks that can be used to obtain estimates of the phase a(h,k,l)
Experimental Phasing Methods MIR - multiple isomorphous replacement - need heavy atom incorporation
MAD - multiple anomalous dispersion- typically done with SeMet replacement MIRAS - multiple isomorphous replacement with anomalous signal SIRAS - single isomorphous replacement with anomalous signal
Computational Methods MR - molecular replacement - need related structure
Direct and Ab Initio methods - not yet useful for most protein crystals
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MAD phasing statistics for the AP-2 a-appendage
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Electron density for the AP-2 appendage
Initial bones trace for the AP-2 appendage
Final trace for the AP-2 appendage
5. Build an atomic model using the electron density map
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The resolution of the electron-density map and the amount of detail that can be seen
Resolution Structural Features Observed
5.0 Å Overall shape of the molecule
3.5 Å Ca trace
3.0 Å Side chains
2.8 Å Carbonyl oxygens (bulges)
2.5 Å Side chain well resolved,
Peptide bond plane resolved
1.5 Å Holes in Phe, Tyr rings
0.8 Å Current limit for best protein
crystals
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6. Validation: How do you know if a crystal structure is right?
The R-factor
R = S(|Fo-Fc|)/S(Fo)
where Fo is the observed structure factor amplitude and Fc is calculated using the atomic model.
R-free
An unbiased, cross-validation of the R-factor. The R-free value is calculated with typically 5-10% of the observed reflections which are set aside from atomic refinement calculations.
Main-chain torsions: the Ramachandran plot
Geometric Distortions in bond lengths and angles
Favorable van der Waals packing interactions
Chemical environment of individual amino acids
Location of insertion and deletion positions in related sequences
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Structure-Based Mutagenesis of the a-appendage
7. Develop structure-based hypothesis
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Selection of E16 specific epitope variants of DIII
Yeast library of DIII variantscreated by error prone PCR
DIII mutations at Ser306, Lys307, ThrE330 and Thr332 significantly diminish E16 binding
Pooled
DIII
mAbs
E16 staining
E -DIII
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GOOD LUCK – Interview season is coming!