Transcript Review Slides

MCB Test 3 Review
M. Alex Miranda
12/17/16
Stem Cell
Biology
Jim Huettner
11/22/2016
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 Cells
Embryonic – from the inner cell mass of preimplantation 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
Inner cell mass
Epiblast: embryo
Hypoblast: yolk sac
http://stemcells.nih.gov/info/scireport/pages/chapter1.aspx
making a knockout mouse
http://en.wikipedia.org/wiki/Knockout_mouse
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?
Two types of ES cells?
“Naïve”
(ICM-like)
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Blastocyst chimera (+)
High cloning efficiency
Short doubling time
Xa Xa
Distal Oct4 enhancer
High Nanog, Klf2/4, Rex1
“Primed”
(Epi-SC)
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Blastocyst chimera (-)
Low cloning efficiency
Long doubling time
Xa Xi
Proximal Oct4 enhancer
Low Nanog, Klf2/4, Rex1
Both types can self renew and give rise to cells from all 3 germ
layers in teratomas or following in vitro differentiation
maintenance of pluripotency - 2
“naïve”
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Positive Regulators
LIF - Stat3
BMP4 - Smad1/5
Wnt (GSK-3 inhibitors)
IGF
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“primed”
TGFb/activin – Smad2/3
FGF2
ERK1/2
Wnt (GSK-3 inhibitors)
IGF
Negative Regulators
• TGFb/activin-Smad2/3
• FGF2
• ERK1/2
• BMP4 – Smad1/5
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 dedifferentiation 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
Removing the bottle neck?
• Rais et al., Nature 502:65-70, 2013 implicate
Mbd3, a component in the NuRD complex that
mediates gene repression via histone deacetylation
and chromatin remodeling.
• Argue that the reprogramming factors recruit both
repressive (Mbd3/NuRD) and de-repressive (Wdr5
and Utx) complexes, and reprogramming only occurs
when the Mbd3/NuRd repression loses.
• Achieve nearly 100% reprogramming within 7 days in
cells with Mbd3 reduced or eliminated.
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
Intracellular Protein Degradation
Chris Weihl MD/PhD
[email protected]
Department of Neurology
Consequence of impaired protein
degradation
• Protein aggregates
• Ubiquitinated inclusions
• Vacuolation
• Damaged organelles
• Secondary impairment in other cellular processes
• Cell Death
• Underlying pathogenesis of degenerative disorders
(neurodegeneration, muscle degeneration, liver
degeneration, lung disease, aging)
Protein Degradation in the Cell
Ub
Autophagy
Nucleus
Aggresome
Ub
UPS
Ub
Ub
Endocytosis
Protein Degradation
 Ubiquitin/Proteasome Pathway
80-90%
Most intracellular proteins
• Lysosomal processes
10-20%
Extracellular proteins
Cell organelles
Some intracellular proteins
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
PROTEASOME COMPONENTS
20S
Proteasome
19S
Particle
ATP
26S
Proteasome
DEUBIQUITINATION
De-ubiquitinating
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
TITLE P AGE I NTRODUCTION
THE P ROCESS
H IS TORIC AL L A N D M A R K S
N O N L I N E A R D EVEL O PM E N TAL P ROGRAM
AND
S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
T HE C ELL B IOLOGY OF A POPTOSIS
T O L IVE IS
TO
DIE – METALLICA (2007)
Paul H. Schlesinger
Department of Cell Biology and Physiology
Office McDonnell 401
Washington University Medical School
[email protected]
December 5, 2016
S CHLESINGER ( WUMS )
A POPTO
D ECEMBER 9, 2014
I NITIATIN
TITLE P AGE I NTRODUCTION
C LAS S IFIC ATION
OF
THE P ROCESS
S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
I NITIATIN
C ELLULAR D EATH
H OW C ELLS A CHIEVE M ORTALITY
Apoptosis – Programmed cell death, controlled part of development
Necrosis – Premature cell death caused by external factors
Autophagy – Degradation of cell components in lysosome. Type of
programmed cell death, separate from apoptosis
Senescence – Cell cycle arrest
S CHLESINGER ( WUMS )
A PO PTO SIS
D ECEMBER 9, 2014
3 / 37
TITLE P AGE I NTRODUCTION
THE P ROCESS
S IGNALING P ATHWAYS BCL-2 P ROTEINS P ORE F ORMING S TRUCTURE
I NITIATIN
MORPHOLOGICAL
A POPTOSIS : M ORPHOLOGY
Morphological Progression
Retain Membrane Barriers
S CHLESINGER ( WUMS )
A PO PTO SIS
D ECEMBER 9, 2014
9 / 37
NMR in biology: Structure, dynamics
and energetics
Gaya Amarasinghe, Ph.D.
Department of Pathology and Immunology
[email protected]
CSRB 7752
• Structure determination by NMR
• NMR relaxation– how to look at
molecular motion (dynamics by
NMR)
• Ligand binding by NMR – Energetics
Protein Structures from an NMR Perspective
Background
–
We are using NMR Information to
“FOLD” the Protein.
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We need to know how this NMR data
relates to a protein structure.
–
We need to know the specific details of
properly folded protein structures to
verify the accuracy of our own
structures.
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We need to know how to determine
what NMR experiments are required.
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We need to know how to use the NMR
data to calculate a protein structure.
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We need to know how to use the
protein structure to understand
biological function
Protein Structures from an NMR Perspective
Analyzing NMR Data is a Non-Trivial Task!
there is an abundance of data that needs to be interpreted
X
Interpreting NMR Data Requires
Making Informed “Guesses” to
Move Toward the “Correct” Fold
Distance from Correct Structure
Not A Direct Path!
Initial rapid convergence to
approximate correct fold
Correct structure
NMR Data Analysis
Iterative “guesses” allow
“correct” fold to emerge
Nuclei are positively charged
many have a spin associated with them.
Moving charge—produces a magnetic field that has a magnetic moment
Spin angular moment
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
Protein Structure Determination
by NMR
•Stage I—Sequence specific resonance
assignment
•State II – Conformational restraints
•Stage III – Calculate and refine structure
Protein Structure Determination
by NMR
•Stage I—Sequence specific resonance
assignment
•State II – Conformational restraints
•Stage III – Calculate and refine structure
NMR Structure Determination
NOE
NOE
- a through space correlation (<5Å)
- distance constraint
4.1Å
2.9Å
Coupling Constant (J)
- through bond correlation
J
NH
CaH
- 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
CaH
D
NH
Protein Structure Determination
by NMR
•Stage I—Sequence specific resonance
assignment
•State II – Conformational restraints
•Stage III – Calculate and refine structure
Protein Structures from an NMR Perspective
What Information Do We Know at the Start of Determining A
Protein Structure By NMR?
Effectively Everything We have Discussed to this Point!
The primary amino acid sequence of the protein of interest.
► All the known properties and geometry associated with each
amino acid and peptide bond within the protein.
► General NMR data and trends for the unstructured (random
coiled) amino acids in the protein.
 The number and location of disulphide bonds.
► Not Necessary  can be deduced from structure.
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
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
Protein crystallography in practice
MCB
15 Dec 2016
Daved H. Fremont
[email protected]
Department of Pathology and Immunology
Washington University School of Medicine
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 luck
The optimum conditions for crystal nucleation are not
necessarily the optimum for diffraction-quality crystal growth
Space Group P21
4 M3 /ASU
diffraction >2.3Å
14.4% Peg6K
NaCacodylate pH 7.0
200mM CaCl2
Space Group C2
2 M3 /ASU
diffraction >2.1Å
18% Peg4K
Malic Acid/Imidazole pH 5.1
100mM CaCl2
Hanging Drop
Sitting drop
Commercial screening kits available from
http://www.hamptonresearch.com;
http://www.emeraldbiostructures.com
Space Group P3121
3 M3 + 3 MCP-1/ASU
diffraction > 2.3Å
18% Peg4K
NaAcetate pH 4.1
100mM MgCl2
3. Collect X-ray diffraction data
Initiate experiments using home-source x-ray generator and detector
Determine liquid nitrogen cryo-protection conditions to reduce crystal decay
While home x-rays are sufficient for some questions, synchrotron radiation is preferred
Anywhere 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
5. Build an atomic model using the electron density map
Electron density for the AP-2 aappendage
Initial bones trace for the AP-2 aappendage
Final trace for the AP-2 aappendage
Low-resolution
At 4-6Å resolution, alpha helices look
like sausages.
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.
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
6. Validation: Mapping of sequence conservation in AP-2 a-subunit appendages
Traub LM, Downs MA, Westrich JL, and Fremont DH: (1999) Crystal structure of the
a-appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly. Proc.
Natl. Acad. Sci. U.S.A. 96:8907-8912.
7. Develop structure-based hypothesis
Structure-Based Mutagenesis of the a-appendage
Traub LM, Downs MA, Westrich JL, and Fremont DH: (1999) Crystal structure of the
a-appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly. Proc.
Natl. Acad. Sci. U.S.A. 96:8907-8912.