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Cell Cycle I
Oh
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What is the basic function of the cell cycle?
• Accurately duplicate the vast amount of DNA in chromosomes
• Segregate the copies precisely into genetically identical daughter cells
Figure 17-2 Molecular Biology of the Cell, 4th Edition
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Figure 17-13 Molecular Biology of the Cell, 4th Edition
What are the phases and checkpoints of the cell cycle?
• G1 – gap between M and S phases
• S – DNA replication
• G2 – gap between S and M phases
• M - mitosis
Checkpoints: G1G2M
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The cell cycle is primarily regulated by
cyclically activated protein kinases
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Cdk activity is regulated by inhibitory phosphorylation and inhibitory proteins
Figure 17-18, 17-19. Molecular Biology of the Cell, 4th Edition
Why is cell cycle progression governed primarily by inhibitory regulation?
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Figure 17-20. Molecular Biology of the Cell, 4th Edition
Cell cycle control depends on cyclical proteolysis
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Mechanisms controlling S-phase initiation
Figure 17-30. Molecular Biology of the Cell, 4th Edition
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DNA damage leads to cell cycle arrest in G1
Figure 17-33. Molecular Biology of the Cell, 4th Edition
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Figure 17-34. Molecular Biology of the Cell, 4th Edition
Overview of the cell cycle control system
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Table 17-2. Molecular Biology of the Cell, 4th Edition
Summary of major cell cycle regulatory proteins
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Extracellular signals dictate the cell cycle
• Mitogens Cell division
• Growth factors Cell growth
• Survival factors --| Apoptosis (tell the cell not to die)
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Figure 17-41. Molecular Biology of the Cell, 4th Edition
Mitogens stimulate cell division
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Figure 17-44. Molecular Biology of the Cell, 4th Edition
Extracellular Growth Factors Stimulate Cell Growth
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Figure 17-47. Molecular Biology of the Cell, 4th Edition
Extracellular Survival Factors Suppress Apoptosis
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Cell Cycle II
Weber
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CANCER IS A DISEASE OF GENETIC MUTATIONS
ACCUMULATION OF MANY MUTATIONS CAUSES CANCER
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YOU MUST REMEMBER THAT ALL MUTATIONS ARE
RANDOMYOU WILL MUTATE DRIVERS AND PASSENGERS
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WHAT MAKES A CANCER CELL A CANCER CELL?
UNLIMITED GROWTH
UNLIMITED MOVEMENT
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THE DEFINITION OF A TUMOR SUPPRESSOR
Classical Features:
1. Loss of function mutations2. Targeted allelic loss- Methylation or Deletion3. Inherited mutations that predispose to cancer4. Somatic mutation in spontaneous tumors5. Ability to inhibit transformed cells in vitro
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ACTIVATING THE p53 RESPONSE
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ACCESSING THE CELL CYCLE MACHINERY
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The Ink4a/Arf Locus
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SELECTIVE PRESSURE THROUGH STRESS
From environment:
-Low oxygen-Low nutrients-Radiation-Ligands
From self:
-Random mutant-ROS-Grow too fast
Dealing with it:
-Arrest/Senesce-Apoptosis
MUTATE
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Not just division… But growth
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Understanding what translation is really all about
Growth signals
PI-3k/mTOR PathwayAnd
Regulators ofRibosome biogenesis 60S 40S
5’
AA
AA
AA
AA
AA
-3’
UTR
UTR
Active translationRibosome biogenesis
rRNA synthesisrRNA processingrRNA export
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PI3K
GFR
Akt
Rheb
Ras
mTOR
Tuberin/Hamartin
PTEN
Neurofibromin
Tuberous sclerosis complex
Neurofibromatosis type 1 (NF1)
Cowden Syndrome Lhermitte-Duclos
disease
S6KeIF4E
NPMARF p68
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Cancer Cell Biology
Stewart
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Murine cells --- two cooperating oncogenes are sufficient to transform
cells
H-rasSV40 LgT
Tumors
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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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The telomere hypothesisT
elo
mer
e L
eng
th
Time
Senescence
Crisis
Stop Rbp53
1 in ~107
Stable telomere maintenance
hTERT
ALT
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SV40ER, TERT, and H-ras cooperate to transform normal human cells
H-ras
TERT
SV40 ER
Hahn et al, 1999
Tumors
T-ag t-ag
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Revised: Functional steps toward cancer
Hanahan and Weinberg, Cell 2011Hanahan and Weinberg, Cell 2000
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Hanahan and Weinberg, Cell 2011
Functional steps toward cancer
Hanahan and Weinberg, Cell 2000
StromaStroma
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null, Volume 144, Issue 5, 2011, 646–674
Is the stroma a participant???
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Tumors are complex “organs”
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Hennighausen and Robinson
The stroma supplies many key signals that lead to duct formation Glibert Smith’s group has used murine neural
stem cells, bone marrow progenitors, cells from testis or human tumor cells and created a new ductal tree in a cleared fatpad! The stroma can dictate differentiation!
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“Humanizing the fatpad allows the outgrowth of human cells
Human fibroblasts
Human epithelial cells
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Barcellos-Hoff, 2000
Irradiated fibroblasts or irradiation of the host stimulates tumor formation
Irradiated fibroblasts
Irradiated host
Normal or preneoplastic mammary cells
Radiated prior to epithelial implantation
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Stroma plays an important role in tumorigenesis
“Normal”Fibroblast
Cancer -associatedFibroblast
“Normal”Fibroblast
Pre-neoplastic Cell
TumorigenicCell
Olumi, 1999
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Formation of a “premetastatic” niche requires the stroma
Sceneay, 2013
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Sceneay, 2013
The stroma impacts tumor cell dormancy
Sceneay, 2013
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null, Volume 144, Issue 5, 2011, 646–674
Stroma contributes to the cancer “continuum”
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A limited cellular lifespan is a potent tumor suppressive mechanism --- in the stroma
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Old stroma promotes tumorigenesis
“Old/Senescent” Fibroblast
“Normal”Fibroblast
Cancer -associatedFibroblast
“Normal”Fibroblast
Pre-neoplastic Cell
Tumorigenic Cell
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SIPS RS Young
SIPSSIPS RS Young
SIPSSIPS RS Young
SIPSSIPS RS Young
Pazolli et al. 2009
Growth factors, ECM, and inflammatory genes are all highly upregulated in senescent fibroblasts
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Box 2. Senescence Stimuli
Replicative senescence (RS) - induced by dysfunctional telomeres
Stress-induced senescence (SIS) – induced by oncogene overexpression, DNA damage, tumor suppressor activation, reactive oxygen species, non physiological culture conditions and other types of stress.
ECM
Young fibroblast
Altered ECM
Senescent fibroblast
Immune cell
Senescent epithelial cell
Cancer cell
Preneoplastic cell
Epithelial cell
Endothelial cell
Senescence evasion
Tumor
Senescence
PremalignantNormal
Stromal Promotion
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Stem Cell Biology
Huettner
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Stem Cells: definition• Self Renewal - undifferentiated cells that can
divide repeatedly while maintaining their undifferentiated state.
• Pluripotency – ability to differentiate into a variety of different cell types
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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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Potency
• Totipotent – able to generate every cell type including extraembryonic tissues
• Pluripotent – able to generate cells from all three embryonic germ layers
• Multipotent – able to generate a variety of cells from a particular somatic structure
• Unipotent – only generate one cell type
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Pluripotency markers• Stage-specific antigens: Anti-SSEA 3 and 4
recognize globo-series gangliosides
• Tra1-60 and Tra1-81: keratin sulfate surface antigens
• Oct3/4, Sox2, Nanog – transcription factors involved with maintaining pluripotency
• Normal karyotype, and pre-X-inactivation?
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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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Reprogramming: somatic cell nuclear transfer
http://www.biotechnologyonline.gov.au/images/contentpages/scnt.gif
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Generating iPS cells• Express transcription factors:
Oct3/4, Sox2, Klf4 and c-Myc 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 Structure and Dynamics
Amarasinghe
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Why use NMR ?
Some proteins do not crystallize (unstructured, multidomain) crystals do not diffract well can not solve the phase problem
Functional differences in crystal vs in solution
can get information about dynamics
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4.1Å4.1Å
2.9Å2.9Å
NOENOE
CCHH
NHNH
NHNH
CCHH
JJ
NOENOE
- a through space correlation (<5Å)- a through space correlation (<5Å)- distance constraint- distance constraint
Coupling Constant (J)Coupling Constant (J)
- through bond correlation- through bond correlation- dihedral angle constraint- dihedral angle constraint
Chemical ShiftChemical Shift
- very sensitive to local changes - very sensitive to local changes in environmentin environment- dihedral angle constraint- dihedral angle constraint
Dipolar coupling constants (D)Dipolar coupling constants (D)
- bond vector orientation relative - bond vector orientation relative to magnetic fieldto magnetic field- alignment with bicelles or viruses- alignment with bicelles or viruses
DD
NMR Structure Determination
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Analysis of the Quality of NMR Protein Structures
With A Structure Calculated From Your NMR Data, How Do You Determine the Accuracy and Quality of the Structure?
• Consistency with Known Protein Structural Parameters bond lengths, bond angles, dihedral angles, VDW interactions, etc
all the structural details discussed at length in the beginning• Consistency with the Experimental DATA
distance constraints, dihedral constraints, RDCs, chemical shifts, coupling constants
all the data used to calculate the structure• Consistency Between Multiple Structures Calculated with the Same Experimental DATA
Overlay of 30 NMR Structures
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Analysis of the Quality of NMR Protein Structures Root-Mean Square Distance (RMSD) Analysis of Protein Structures
• A very common approach to asses the quality of NMR structures and to determine the relative difference between structures is to calculate an rmsd
an rmsd is a measure of the distance separation between equivalent atoms
two identical structures will have an rmsd of 0Å the larger the rmsd the more dissimilar the structures
0.43 ± 0.06 Å for the backbone atoms 0.81 ± 0.09 Å for all atoms
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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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Structural Biology: X-ray Crystallography
Fremont
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A 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 diffusionSitting drop, dialysis, or under oilMacro-seeding or micro-seedingSparse 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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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 (h,k,l)
Experimental Phasing MethodsMIR - multiple isomorphous replacement - need heavy atom incorporation
MAD - multiple anomalous dispersion- typically done with SeMet replacementMIRAS - multiple isomorphous replacement with anomalous signalSIRAS - single isomorphous replacement with anomalous signal
Computational MethodsMR - 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 -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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Medium resolution
~3Å data is good enough to see the backbone with space in between.
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Holes in rings are a good thing
Seeing a hole in a tyrosine or phenylalanine ring is universally accepted as proof of good phases. You need at least 2Å data.
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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 = (|Fo-Fc|)/(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 -appendage
7. Develop structure-based hypothesis