MCB Exam III Review

Ji Woong ParkDecember 13th, 2014

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.

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

Telomere Function

distinguishes between the chromosome end and a double strand break

protects the chromosome from end-to-end fusions

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

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

The telomere hypothesisTe

lom

ere

Len

gth

Time

StopRbp53 Senescence

The telomere hypothesisTe

lom

ere

Len

gth

Time

Continued proliferationRbp53

Crisis

telomere dysfunctiongenetic catastrophecell death

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

The telomere hypothesisTe

lom

ere

Len

gth

Time

Senescence

Crisis

Stop Rbp53

1 in ~107

Stable telomere maintenance

hTERT

ALT

The telomere hypothesisTe

lom

ere

Len

gth

Time

Crisis

Stop

1 in ~107

hTERT

ALT

ALT

Telo

mer

ase

+

Telomerase allows ALT cells to form tumors

H-ras

GM847

TUMORS

hTERT

Lacks telomeraseExpresses SV40 Early regionImmortal

Stewart et al 2002

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

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

For additional reading (highly recommend)

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)

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.

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

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 Degradation in the Cell

UPS

Aggresome

Autophagy

Endocytosis

Nucleus

Ub

Ub

Ub

Ub

Protein Degradation Ubiquitin/Proteasome Pathway

80-90%Most intracellular proteins

• Lysosomal processes10-20%

Extracellular proteinsCell organellesSome intracellular proteins

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

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

The proteasomal DUB Usp14 impairs protein degradation

Lee, BH et alNature 467:179-842010

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)

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

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

Protein Structures from an NMR Perspective

Dis

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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

Protein Structure Determination by NMR

•Stage I—Sequence specific resonance assignment

•State II – Conformational restraints

•Stage III – Calculate and refine structure

Why use deuteration?

• What are the advantages?

• What are the disadvantages?

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

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

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

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

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

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

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 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

MAD phasing statistics for the AP-2 a-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

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 = 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

Structure-Based Mutagenesis of the a-appendage

7. Develop structure-based hypothesis

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

GOOD LUCK – Interview season is coming!