Cell Cycle I Oh. What is the basic function of the cell cycle? Accurately duplicate the vast amount of DNA in chromosomes Segregate the copies precisely

Cell Cycle I

Oh

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

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

The cell cycle is primarily regulated by

cyclically activated protein kinases

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?

Figure 17-20. Molecular Biology of the Cell, 4th Edition

Cell cycle control depends on cyclical proteolysis

Mechanisms controlling S-phase initiation


DNA damage leads to cell cycle arrest in G1



Overview of the cell cycle control system

Table 17-2. Molecular Biology of the Cell, 4th Edition

Summary of major cell cycle regulatory proteins

Extracellular signals dictate the cell cycle

• Mitogens Cell division

• Growth factors Cell growth

• Survival factors --| Apoptosis (tell the cell not to die)


Mitogens stimulate cell division


Extracellular Growth Factors Stimulate Cell Growth


Extracellular Survival Factors Suppress Apoptosis

Cell Cycle II

Weber

CANCER IS A DISEASE OF GENETIC MUTATIONS

ACCUMULATION OF MANY MUTATIONS CAUSES CANCER

YOU MUST REMEMBER THAT ALL MUTATIONS ARE

RANDOMYOU WILL MUTATE DRIVERS AND PASSENGERS

WHAT MAKES A CANCER CELL A CANCER CELL?

UNLIMITED GROWTH

UNLIMITED MOVEMENT

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

ACTIVATING THE p53 RESPONSE

ACCESSING THE CELL CYCLE MACHINERY

The Ink4a/Arf Locus

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

Not just division… But growth

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

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

Cancer Cell Biology

Stewart

Murine cells --- two cooperating oncogenes are sufficient to transform

cells

H-rasSV40 LgT

Tumors

Telomere Function

distinguishes between the chromosome end and a double strand break

protects the chromosome from end-to-end fusions

The telomere hypothesisT

elo

mer

e L

eng

th

Time

Senescence

Crisis

Stop Rbp53

1 in ~107

Stable telomere maintenance

hTERT

ALT

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

Revised: Functional steps toward cancer

Hanahan and Weinberg, Cell 2011Hanahan and Weinberg, Cell 2000

Hanahan and Weinberg, Cell 2011

Functional steps toward cancer

Hanahan and Weinberg, Cell 2000

StromaStroma

null, Volume 144, Issue 5, 2011, 646–674

Is the stroma a participant???

Tumors are complex “organs”

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!

“Humanizing the fatpad allows the outgrowth of human cells

Human fibroblasts

Human epithelial cells

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

Stroma plays an important role in tumorigenesis

“Normal”Fibroblast

Cancer -associatedFibroblast


Pre-neoplastic Cell

TumorigenicCell

Olumi, 1999

Formation of a “premetastatic” niche requires the stroma

Sceneay, 2013

Sceneay, 2013

The stroma impacts tumor cell dormancy

Sceneay, 2013

null, Volume 144, Issue 5, 2011, 646–674

Stroma contributes to the cancer “continuum”

A limited cellular lifespan is a potent tumor suppressive mechanism --- in the stroma

Old stroma promotes tumorigenesis

“Old/Senescent” Fibroblast


Cancer -associatedFibroblast


Pre-neoplastic Cell

Tumorigenic Cell

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

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

Stem Cell Biology

Huettner

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

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)

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

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?

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.

Reprogramming: somatic cell nuclear transfer

http://www.biotechnologyonline.gov.au/images/contentpages/scnt.gif

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

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

Protein Structure and Dynamics

Amarasinghe

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

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

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

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

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

Structural Biology: X-ray Crystallography

Fremont

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

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

http://www.protein-solutions.com/

http://www-structure.llnl.gov/cd/cdtutorial.htm

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

http://www.hamptonresearch.com/

http://www.emeraldbiostructures.com/

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

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

MAD phasing statistics for the AP-2 -appendage

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

Medium resolution

~3Å data is good enough to see the backbone with space in between.

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.

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

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

Structure-Based Mutagenesis of the -appendage

7. Develop structure-based hypothesis

Documents

Cell Cycle I Oh. What is the basic function of the cell cycle? Accurately duplicate the vast amount of DNA in chromosomes Segregate the copies precisely