LECTURE : STRUCTURE OF PROTEIN
Ramachandran plot
Glycine can adapt many more positions in the plot, and many positions in the backbone
Proline is much more restricted, Ramachandran plot very restricted.
It restricts amino acid on N-terminal residue of proline, the residue is also restricted due to proline.
On alpha helix, 3.6 amino acids (5.4Angstrom) between a turn of helix; hydrogen bonds between carboxyl oxygen and amide's proton, can be right or left handed
views of alpha helix
ribbon representation, (D) - representation of DNA by van der Waals surface
Beta pleated sheet
anti-parallel N->C, hydrogen bonding with C <- N terminus below
parallel - all peptides pointing to one direction,
these forms have different hydrogen bonding
In antiparallel, each amino acid bound to a single amino acid on an adjacent strand
In parallel, each amino acid on one strand with two different amino acids(two residues apart) on adjacent strand
Beta turns, tight turns with Gly or Pro
type I - larger residues than type II
type II ???
distance between amino acid in beta sheet 3.5 Angstrom, compared to 1.5 anstrom of alpha helix
Beta sheet, most commonly found in active sites??
Structure class of protein (tertiary structure)
Globular proteins - contain all secondary structure elements, high functional diversity
enzymes, recepters, etc.
Fibrous proteins - rather specialized for structural roles
all alpha-helix structures: e.g. collagen, vs. all beta-sheet
Collagen - proline helix: Gly-X-Pro(or OH-Pro); left handed structure(going toward viewer on clockwise rotation when viewed from top), 3 aa/turn; triple helices come together to form a collagen, proline on the exterior, Gly on the interior
Different amino acid show propensity for different secondary structure(alpha vs beta), so can predict secondary structure from primary structure, but tertiary structure still needs to be determined experimentally
Enormous diversity of protein globular structures, mixture of alpha helices and beta sheets
Rules of 3' structure
- Hydrophobic side chains mostly inside
- Multiple 2' strucutres may exist in a single protein
- Pro, Gly often located at turns , outside??
- Compact interiors (very little room even for H2O)
- Functional (active) sites involve conserved side chains often far apart in the 1' sequence - but can come together in 3' structure
- S-S bridges greatly stabilize 3' structures
Quarternary structure: symmetric dimers(e.g. Hb) to large complexes (RNA polymerase)
Structures determined by
1) X-ray crystallography
- atomic resolution images of macromolecules
protein crystal that is purified (1mg of protein sufficient), crystals diffract X-ray, plot electron density map from diffraction intensities(image of phases)
how to get phases diffraction data (determined by how much the structure is known already):
1) multiple isomorphous replacement (de novo strucutre determination)
2) molecular replacement (requires related structure)
3) differences fourier syntehsis (requires very related structure, same crystal packing, useful for drug-protein complex, rapid)
-> fit model -> refine structure based on known stereochemistry
protein crystals
- very large solvent channels, can be up to 75% solvent
- many enzymes crystalled in different solvent and form, but structure remain mostly identical
- enzymes in families with similar amino acid sequences have structures very similar
- In many enzymes, the crystal retains activity, the activity of enzyme in solution is same as in crystal, e.g. ribonuclease, chymotrypsin, DNA polymerase
2) NMR
- measure proton chemical shift that allow constrain positions of proton -> construct the most probable model
3) Ribbon representation : Alpha helix (as helix), beta sheet represented by arrow, N -> C
Protein misfolding disease
e.g. - beta-amyloid fibril misfold , Alzheimer, Transmissible spongiform encephalopathy, cancers associated with protein overproduction
Chymotrypsin
2 domain structure, active site residues can be far apart based on 1' sequence, (H57, S195, D102), come together to form active site
Pancreatic Ribonuclease A(digestive enzyme that cleaves RNA)
- location of bound protein determines active site, essential catalytic side chains adjacent to active site (H119, H12)
HIV-1 protease
- dimer - 99 a.a. flap opens up, wraps around a protein substrate, for cleavage
- HIV inhibitor drugs mimic a protein chian, binding to enzyme like protein chains do, but they are more stable than protein chain and thus HIV-1 protease cannot cleave them, and the inhibitor stays lodged to the active site.
DNA polymerase in action (structure can move around a lot)
4 crystal structures in different states
MutSalpha
heterodimer,
find mismatch paired DNA, recruit other proteins to repair
sites that hydrolyzes ATP, send signal down to specific piece of DNA, spheres represent mutation -> HNPCC
Importance of cofactors in protein? - able to execute chemical reactions that cannot be performed by standard amino acids; enzyme w/o its cofactor = apoenzyme, enzyme w/ its cofactor = holoenzyme
- can be metals or coenzymes(small organic molecules), tightly bound coenzyme are called prosthetic group
LECTURE: CELLS AS UNITS OF STRUCTURE
Tenets of cell by Schleiden and Schwann
1) cells units of structure, physiology and organization of living things
2) cells retain a dual existence as a distinct entity and a building block in the construction of organism
Virchow
every animal as sum of vital units
Omnis cellula e cellula
Diversity of cell size, shape and function
sperm cell, neuron, photoreceptor cell, hair cell
Light microscope resolution limit 1/2 of light wavelength = 0.275 micron
Units of size - cell structure
cm - visible to eye
mm - very large cells
µm - cells, nucleus
nm - organelles
angstrom - molecules, atom
special stain for smaller structure, population of organelles/small structure
time lapse video microscopy - cells in action/migration/cell-cell interaction
Electron microscope
- resolution is 1/100000 wavelength of light
- common resolution limit is 10 angstrom
- advanced to single atom resolution, or sub-atomic resolution
- limitation: has to chemically fix the cell
How do we study cell function?
Cell fractionation - allows identify structure and function
Cell architecture
- metabolic compartmentalization - allows diversity of regulation that prokaryote cells can't do
1) nucleus
- segregate gene transcription and RNA translation
- outer and inner nuclear envelopes (double layer)
- nuclear pore core complex - regulate information access to chromatin
- heterochromatin(tightly compact, transcriptionally silent), euchromatin (dispersed, transcriptionally active)
- endoplasmic reticulum - physical continuation of outer nuclear envelope
- nucleolus - functional compartment(not membrane bound), site of ribosomal RNA synthesis and ribosome biogenesis
- altered nuclear morphology, multiple nucleoli, dispersed chromatin(euchromatin abundant, heterochromatin sparse) -> transcriptionally active
Nuclear pore complex
- gateway of information transfer
- ubiquitous structure present in all eukaryotic cells
- located at sites where inner and outer envelopes fuse
- two prominent structures: nuclear and cytoplasmic rings, central spokes
- 8 fold symmetry
- very complex/large: 100-200 proteins, 1200 A in diameter, 500 A in width
- cytoplasmic side has filaments for binding, nucleoplasmic side has basket for docking site for components that are exiting the nucleus
- proteins that line the nuclear pore complex - native unfolded proteins - very relaxed protein structures, very interactive, adhesive scaffold - to allow specific transport
NPC-mediated nuclear transport
- transport thorugh NPC by
- simple passive diffusion (20-40kDa)
- active transport (> 40 kDa, require ATP/GTP, require nuclear localization signal(NLS) - 8-10 amino acid long positive charged amino acid, can reside in any exposed part of protein)
- mediate bidirection transport and unidirectional mRNA transport
Nuclear import pathway
- paticular signal in protein that indicates that it be traffcked to certain location
- importin alpha binds to substrate, and bind to importin-beta, importin beta bind to filament, guide substrate cargo into the pore into nucleoplasm and then dissociate
Role of GTP binding protein
- different conformation based on nucleotide thats bound, allow different activity
- Ran-GAP(GTPase activating protein) -> GTP to GDP in cytoplasm
- in cytosol, Ran mostly in GDP bound state,
- in nucleus, Ran mostly in GTP bound state
- 1) Ran GTP binds to substrate, 2) converted to GDP+substrate-alpha+beta
- this complex 3) enters nucleoplasm, 4) where GDP -> GTP by guanine nucleotide exchange, 5) which releases alpha and substrate, while 6) GTP-beta transpored out to cytoplasm to find another substrate-alpha complex
two states - two conformations - two distinct activities in RanGTP binding protein
RanGDP in cytoplasm, RanGTP in nucleus
Nuclear Export pathway
- identify by cluster of leucine residues (hydrophobic signal), unlike import where its identified by cluster of positive charges
- exportin - binds GTP in nucleus, enhance the affinity for its cargo
- cargo-exportin-GTP compelx binds to FG(Phe-Gly) repeats, facilitate its diffusion across the pore
- RanGAP in cytoplasm cause GTP hydrolysis -> decrease affinity betwen cargo and exportin -> release cargo into cytoplasm
Nuclear lamina
- in inner nuclear side, dense fibrous network composed of three lamins A, B, C
- phosphorylation of lamin -> disassembly of lamin / nuclear envelope
- lamin B connected to inner membrane via p58; A and C are splicing products of the same gene
- laminopathy - disease due to mutation of lamin A/C gene
-e.g. Emery-Dreifuss muscular dystrophy type 2, familiar partial lipodystrophy, limb girdle muscular dystrophy type 1B, Charcot-Marie-Tooth disorder type 2B1, mandibuloacral dysplasia, Hutchinson-Gilford(childhood progeria syndrome)
2) endoplasmic reticulum
- continuous with outer nuclear envelope
- two types: rough and smooth
- RER - granular rough surface, ribosomes, syntesis of secretory and membrane proteins
- SER - highly rich in liver, function in detoxification
- serves as a Ca2+ storage site
- can comprise of 70% of surface area of cell ???
- on outer nuclear envelope with ER, nuclear pore complex excluded from endoplasmic reticulum
3) Golgi apparatus
- modification of secretory membrane proteins (particularly sugar/oligosaccharide modification)
- cis side - receiving side/ entrance side from ER
- trans side - shipping side
4) Lysosomes
- degradative function
- serves as terminal trafficking site for products of receptor-mediated endocytosis
- highly acidic compartment
- also degrade old organelles
- particularly abundant in antigen presenting cells
5) peroxisome
- single membrane organelle
- beta oxidation, primarily for lipid metabolism
- e.g. bile acid synthesis, cholesterol synthesis, plasmalogen synthesis, amino acid and purine metabolism
- many associated genetic defects
6) Mitochondria
- abundant organelles of cell,
- largest organelle
- primary source of ATP, functions in oxidative metabolism
- e.g. heme and iron sulfur center biosyntehsis and modulating Ca2+ levels
- associated with e.g. neurodegenerative, ischemia-perfusion injury, diabetes and aging
LECTURE: MEMBRANES AND MITOCHONDRIA
ER can pinch off vesicle, to fuse with Golgi apparatus, which secretes secretory vesicle to fuse with cell membrane to exocytosis
Membrane fusion in exocytosis and endocytosis - material can enter the cell without crossing lipid bilayer
- gives rise to
- 2 spaces in cell: extracellular space around the cell(in continuity with space in secretory vesicle, golgi apparatus, RER) and cytoplasmic space(and nucleoplasm).
Membrane functions
- compartmentalize cell
- regulate flow of material in/out of cell
- excitable membranes = basis for nerve conductions, muscle contractions
- site for many enzyme rxn -if enzymes are membrane bound in a closed area, the rxn can be much more efficient than if the enzymes are not bound and just floating around
- regulate cell shape, motility through interactions with cytoskeleton
- tissue formation - rarely cells live alone; role of membrane junctions - cells join together by membrane junction
- interaction of cell with environment; sensor of external signals -> amplifed/transduced
- signal transduction
Membrane composition
- Lipid 20-40% - major component
- Proteins 30-50% - major component
- Carbohydrates 0-10%
- Water 10-20%
- work together as a functional unit
Three primary types of membrane lipid
1) phospholipids (PL)
- phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine
2) glycolipids (G)
3) cholesterol (C)
Composition of different organelle membranes
inner mitochondrial membrane - almost all PL
RER -> Golgi -> plasma membrane (amount of cholesterol increases - rigidity)
Almost all G and majority of cell's C in plasma membrane
1) phospholipids (PL)
All the lipids are amphipathic (LOOK AT THE DIAGRAM)
Glycerol, phosphate, alcohols are polar part of the molecule
fatty acids - hydrophobic part of molecule
Most common type of PL in membrane has saturated FA on 1 position, and unsaturated FA on 2 position; huge diversity in hydrophobic chain composition and head composition
2) glycolipids (G)
Glycolipids(G) have different backbone, most of them have sphingosine backbone
backbone types; G is a type of sphingolipid
- sphingosine - acyl chain
- glycosphingolipids - sphingosine backbone with FA chain and sugar
- sphingomyelin - sphingosine backbone with FA chain and phosphate and alcohol(choline or ethanolamine); both a PL and a sphingolipid
- glycosphingolipids and sphingomyelin have more saturated fatty acid chain than membrane phospholipids
3) cholesterol (C)
amphipathic, but the hydrophilic part is just an OH group, hydrophobic part has rings
very rigid molecule, decrease fluidity of membrane
membrane rigidity decreased by double bond(unsat), and increases fluidity
Why these types of molecules to be in cell membrane?
- these three classes comprise 98-99% of lipids in membranes
- they naturally only form bilayers, b/c they are more cylindrical shaped
- detergents, they are inverted cone shape, so they form micelle - not useful for membrane
lipid bilayers are liquid crystals
- very rapid lateral diffusion (10^7 times per sec)
- very rare flip-flop (once per month? very crystal)
permeability barrier
- hydrophobic molecule, O2, N2 go through
- small molecules like H20, urea, glycerol and CO2 will cross
- ions and large uncharged molecules e.g. glucose will not
Roles of membrane lipid
1. structural - lipid bilayer is a core of all biological molecule
2. bilayer hydrocarbon core is thin only 3 nm, semi-permeability barrier
3. regulate membrane fluidity, flexibility (add cholesterol for toughness, plasma membrane has many, while internal organelle has little)
4. role in membrane fusion, different lipid fuse at different rates
5. function/location of membrane protein
- matrix for membrane bound enzymes (certain lipid type required for activity of specific enzyme)
- covalently anchor specific proteins to bilayer(phosphatidylinositol can attach via sugar linkage, protein to lipid bilayer)
- target peripheral proteins to proper membrane organelles (phosphatidylinositol)
- sequester specific proteins in membrane domains or rafts (
cholesterol likes to be next to saturated chains(namely sphingomyelin) -> create little patch in membrane(domain or raft) -> platform for signal transduction pathway
6. interaction with extracellular matrix and other cells (G)
7. specific lipids can be source of second messenger (phosphatidylinositol bisphosphate)
Membrane protein types
1) transporters
- passive
- active Na K ATPase
2) anchors
- e.g. spectrin in RBC
- cytoplasmic surface, form membrane cytoskeleton
3) receptor
4) enzyme
Mitochondria
- cristae - infolding
- intermembrane space
- matrix space
Flow of reactants in mitochondrion
- citric acid cycle -> CO2, NADH (-> electron to membrane proteins, -> pH gradient -> hydrogen ion move the pump to convert ADP -> ATP)
Electron transport system
- three complexes
- NADH dehydrogenase complex (complex I) - NADH donate e- to the complex, then to ubiquinone, then diffuse to
- b-c1 complex (complex III) -> cytochrome c, diffuses to
- cytochrome oxidase complex (IV)
- hydrogen ion move out as e- go through these complexes
- then Hydrogen ions move through ATP synthatase which produce ATP
- outer membrane has protein channels called porins, which allow
- all the ion/ATP/ADP, etc could go passively across outer membrane
- however ATP/ADP can get into inner mitochondrion only by active process, energy from H+ gradient
General features of membrane structure critical for functions of mitochondria
IMM
1. fluid lipid bilayer allows allosteric changes in protein and lateral diffusion of carriers in electron transport chain
2. e- transfer, both intrinsic (cytochrome oxidase) and extrinsic (cytochrome c) proteins play roles
3. specific lipids (cardiolipin) necessary for cytochrome c oxidase function
4. lipid bilayer forms permeability barrier to H+
5. transporter proteins provide for passage of ATP, ADP, other molecules across IMM
OMM
1. membrane channels(porins) allow ATP to passively diffuse
2. porins small enough pore to keep cytochrome c in intermembrane space;
formation of channels(Bak/Bax proteins bind to membrane) that allow cytochrome c to exit -> kep step in apoptosis
LECTURE : SPECIAL CASE OF MEMBRANE PROTEINS
Two classes of membranes
Peripheral
- not embedded in the bilayer but stuck on the surface of bilayer
- 1/3 of the total membrane protein
- remove with treatment that doesn't disrupt bilayer, that uses polar interaction
e.g. salt, chelator, urea - bind to surface protein
- they don't have bound lipids
- no exposed hydrophobic surface
Integral
- 2/3 of total membrane protein
- can remove by destroying the bilayer (detergents, organic solvents)
detergents - have both hydrophobic and hydrophilic region, but they have rather large hydrophilic region(polar head group), relatively small hydrophobic tail(typically only one, not two) : e.g. octyl-glucoside (non-ionic), sodium dodecylsulfate (SDS, ionic)
- detergents incompatible with packing into bilayers
- form micelle - at crtically micelle concentration
- when adding detergent to bilayer, initially detergents integrate into bilayer, but it becomes unstable as more added, form mixed micelles - each micelle contains one membrane molecule, or one membrane protein
- membrane proteins can remain active if non-ionic detergent used
- protein hydrophobic -> can span the membrane
- have bound lipid - help contribute to proper folding and anchoring in the bilayer
- aggregate in hydrophilic environment
Purification of membrane proteins in non-detergents
- can use same methods as chromatography of soluble proteins, however the detergent should always be present, otherwise hydrophobic part of protein will fold itself and aggregate
- reconstitution - remove excess detergent in the presence of phospholipids, remove the protein from micelle into bilayer/membrane like structure
characterization of membrane protein
- crystallography most informative
Membrane protein functions
- enzymes
- lipid metabolism and glycosylation
- electron transport complexes
- ATP synthesis complex
- receptors
- signaling - G protein linked, kinase activating
- protein uptake - LDL, transferrin
- antigen presentation - MHC complex
- small molecules transporters -glucose, drug, CFTR
- channels - water(aquaporin), ions
Covalent modification of membrane proteins with lipids
- acylation (fatty acid attached to membrane protein)
built from 2 carbon units
can be attached to N, O, or S group
e.g. N-terminal myristate, palmitate (O, S)
- prenylation (15, 20 carbons attached to S of Cysteine)
built from 5 carbon units
PI(phosphatidylinositol)-glycan linked protein
- behave like integral protein but functionally they are peripheral
- structurally protein is actually peripheral on the outside, but has hydrophobic components that links it to the surface
- present in all eukaryotic cells
- e.g. defective in paroxysmal nocrutrnal hemoglobinuria(PNH), particularly abundant on malarial parasites
Integral membrane proteins often attached to membrane through hydrophobic transmembrane helices
- typically alpha helices (20-25 residues long)
- hydrophobic residues in transbilayer region
- often predicted from their 1' sequence(hydropathy analysis)
add up + for hydrophobic, - for hydrophilic side chains, add them up in segments of 25; if high number, more likely to be transmembrane segment
# of transmembrane helices in membrane protein
- one : LDL receptor, Golgi, glycosyl transferase
- seven: G protein linked receptors (-800 in humans)
- 10-12: transport protein (glucose transporter)
X-ray structure of membrane proteins
hydropathy prediction useful
- bovine rhodopsin
- beta adrenergic receptor
- bacteriorhodopsin
- human aquaporin - defective in nephrogenic diapbetic inspidius
- bacterial K+ channel
hydropathy prediction misleading
- prostaglandin H2 synthase
- E.coli membrane porin - transmembrane region is beta-sheet instead of alpha helix
membrane protein vs soluble protein
- membrane protein (integral) typically have hydrophobic outside in the middle, while soluble have hydrophobic on the inside
- membrane protein typically have channels(hydrophilic), while soluble proteins don't have channels
- peripheral protein more likely to be hydrophilic, and hydrophobic residue buried inside
LECTURE : HEMOGLOBIN
Globin genes
types of Hb
A2 - minor Hb, delta chain replaces beta chain ; 1.5-3.5%
F - fetal Hb, in adults usually a sign of something
S - sickle cell Hb, mutated beta chain
pathologic Hb
C - Hemoglobin C results from a mutation in the beta globin gene and is the predominant hemoglobin found in people with hemoglobin C disease (a2bC2). Hemoglobin C disease is relatively benign, producing a mild hemolytic anemia and splenomegaly. Hemoglobin C trait is benign.
E - This variant results from a mutation in the hemoglobin beta chain. People with hemoglobin E disease have a mild hemolytic anemia and mild splenomegaly. Hemoglobin E trait is benign. Hemoglobin E is extremely common in S.E. Asia and in some areas equals hemoglobin A in frequency
H - Hemoglobin H is a tetramer composed of four beta globin chains. Lacks three alpha-globin genes. Hemoglobin H occurs only with extreme limitation of alpha chain availability. Hemoglobin H forms in people with three-gene alpha thalassemia as well as in people with the combination of two-gene deletion alpha thalassemia and hemoglobin Constant Spring.
Barts. Hemoglobin Barts develops in fetuses with four-gene deletion alpha thalassemia. These molecules transport oxygen poorly. Most individuals with four-gene deletion thalassemia and consequent hemoglobin Barts die in utero (hydrops fetalis).
thalassemia(deficiency of alpha or beta chain) AB+, beta-thalassemia heterozygote lacks a beta chain
- in alpha thalassemia, alpha globin chain is underproduced. No compensatory increase of any other chains
- beta thalassemia minor(heterozygote), beta chain underproduced
- beta thalassemia major(homozygote), beta chain absent
- compensatory increase of HbF, but not adequate
F - alpha2gamma2 dimer
A2 - alpha2delta2 dimer
A - alpha2beta2 dimer
myoglobin - monomeric protein
tissue = 20 mmHg - partial pressure of oxygen in tissues
Hb cooperativity - larger fraction of total bound Hb can be released than if dissociation was not sigmoid
T state vs R state
T state (deoxy-Hb)
- no oxygen, so attaching O group initially can be difficult, but if one O bound, it makes the rest better O binders (K2 > K1)
R state (Oxy-Hb)
- all four sites occupied by oxygen
active site of hemoglobin is heme cofactor - Fe-protoporphyrin IX
- rings part hydrophobic
- sits between helices in the middle of the protein
- carboxyl groups stick out on the surface
- Fe can bind to 6, among which it binds to N on heme cofactor, and remaining two are
- bound by His residue
- one binding site for O2
=> once Fe fully bound, Fe slightly moves into plane with the tetrapyrole ring, and His residue follows => His residue moves alpha helix a little bit => change interaction at the alpha beta interface (alpha1beta1-alpha2beta2)
- binding of O2 -> rotates alpha/beta dimer by 15 degrees
allosteric effector
- homotropic effector (effector is same thing as whose function/binding affected)
Oxygen
- heterotropic effector
2,3 -BPG (metabolite of glucose), equal amount of BPG in RBC as Hb
CO2
proton (pH)
2,3-BPG binds between beta subunits where they have basic residues as binding site
- stabilizes T state, favoring release of O2
- 2,3-BPG does not bind to R state
- HbF missing on Histidine residues(2 out of 4) of this site, thus it doesn't bind 2,3-BPG
-> thus it is on higher affinity binding state for O2 than maternal
-> O2 tends to flow from materal oxy-Hb to fetal deoxy-Hb; but this only works when 2,3-BPG is present, w/o 2,3-BPG HbF no different from HbA in binding affinity
low pH and increased CO2 shift ODC to the right, Bohr effect
CO2 directly interacts with Hb amino acid N terminal -> carbamate -> alters alpha/beta subunit interactions such that low affinity T state is favored
pH
- Three amino acid residues, Asp94, His146 (important H bond btwn Asp and His), Lys40 form hydrogen bonds, when these hydrogen bonds intact, Hb in T state, favors release of O2
- decreased pH makes His146 more positive, thus stronger hydrogen bond interaction with Asp94
SUMMARY
- 2,3-BPG, CO2, pH, key regulators, work at different sites
- increased 2,3-BPG, H+ and CO2 favor T state
- effects can be additive since different sites involved
CO bind 210 times more affinity than O2 on Hb
CN,
- does not bind to iron in 2+ state, instead
- bind to iron sites involved in respiration in mitochondria - a.k.a. cytochrome oxidases
- treatment
sodium nitrite/amyl nitrite
convert Hb to Met-Hb (Fe3+) - 10-15% => CN can bind to Met-Hb
better than have CN bound in mitochondria, give kidney time to excrete CN
thiosulfate - convert CN to SCN(inert) -> body can excrete
LECTURE : SICKLE CELL
sickle cell disease
- an evolutionary response to malaria
- probably evolved 14,000 yrs ago when humans began to live settled life in tropics
- hx
- 1910 - first clinical description (Herrick) - sickle shaped cell in blood
- 1927 - Sickling phenomenon noted (Gillepsie and Hahn) - deprived of O2, cells sickle, did not distinguish homozygote and heterozygote
- 1949 - differences by electrophoresis noted - identification of HbS status by electrophoresis (AA vs. AS vs. SS), genetics unraveled (Itano and Neel)
- 1956 - single amino acid difference in beta chain (Ingram)
- advantage in fighting malaria, only in heterozygotes(AS), homozygote(SS) survival decreases due to sickle cell disease (Glu -> Val)
- different haplotypes of HbS arose from different parts of the world where malaria was prevalent
- molecular alteration
- sickle change occurs on part of beta chain that's facing outward
- as HbS molecule deoxygenated,
unpolymerized HbS -> nucleation(small group line up) -> random rods(elongation into long lines) -> parallel rods
=> due to Val in HbS able to interact with neighboring Hb's beta chains' Ala 70, Phe 85, Leu 88 ; if HbA is present, it will get into the chain and stop the chain from forming, however, if only HbS present, these chains will continue to form long paracrystals - basis for pathophys of sickle cell dz
- paracrystals
cause of shape change
Oxygen depleted then cells sickle; if O2 added back RBC returns to normal shape b/c oxy-Hb will have conformation where Val group contact point is covered
- however, if O2 depletion prolonged, RBC will retain sickle shape even if Hb no longer retains sickle form
- clinical consequence
1) effects of hemolysis
- anemia
- normal RBC lifespan 100(SD30 days), while sickle RBC only lasts 15-20 days
- diminished O2 supply, work capacity
- compensatory overwork of heart which can lead to failure
- compensatory overgrowth of bone marrow
- needs vitamin supplement (folic acid)
- affect bone strength
- hemolysis results from cell deformation
2) changes RBC membrane
- results in adhesion
with other RBC-> phagocytosis
with endothelium -> vascular obstruction
- as cells pass from arteriole -> capillary -> venule, O2 removed and cells sickle by the time they are in venous sytem
3) vascular obstruction
- cause pain in areas where oxygen low due to vascular obstruction
- once obstruction then pain, it resolves in 5-7 days
- loss of spleen
- due to sickle cell clotting and decreased O2 supply, becomes scar tissue
- spleen is an immune defense from bacteria, loss of spleen results in 25-30% of sickle cell pts die before 5 yrs age
- bleeding from kidney and diminution of kidney function
- changes in eyes
- small areas of loss of tissue and function
- increased intracellular viscosity, leads to increased blood viscosity
components of whole blood viscosity
- plasma viscosity - minor
- proportion of red cells - major
- intrinsic red cell viscosity - major
increased viscosity
- larger vessels may be affected, RBC need not be sickle shaped, but overall increase in intrinsic RBC viscosity may cause a group of these cells to sludge
- pain
- obstruction of larger vessels
- poor circulation to the lungs - may cause pneumonia or acute chest syndrome (the most common cause of death in sickle cell disease) - treatment is to replace the pt's blood with normal blood (transfusion)
- strokes - major blood vessel of brain can become obstructed
- loss of blood to bone/joint -> acute vascular necrosis, hip arthritis
toll of sickle cell dz
- loss of function of organs(brain, lung, liver, spleen, kidney, heart, etc)
- difficulty in social function
- 60% do not enter work force
- diffculty maintaining a job
- medical cost, $26,000/yr
- shortened lifespan (50-55 yrs)
treatment
- pain management
- drugs that reduce sickling (e.g. hydroxyurea)
- bone marrow transplantation
-
LECTURE : IRON HOMEOSTASIS
ferritin - holds up to 4500 iron atoms, polymer of 24 polypeptide subunits, stores iron within cells
transferrin = Fe3+
DMT-1 requires proton for co-transport
iron uptake during erythropoiesis - endocytosis of transferrin-iron by RBC
- transferrin-iron complex binds to receptor on cell membrane
- endocytosis, and endosome becomes more acidic by pumping in more proton
- DMT1 releases iron, ferritin released by exocytosis, re-used for iron uptake
macrophage iron transport
- macrophage engulf RBC, iron released from macrophage via ferroportin, which is bound to transferrin
iron transport
duodenum - DMT1 - iron uptake, ferroportin - iron release to blood stream
erythroid precursor - DMT1, transferrin receptor(TFR) - for uptake of iron
liver - macrophage, phagocytosis of RBC - iron uptake, storage
iron regulatory element(IRE) - function depends on where it is found, forms stem loop structure
iron regulatory protein(IRP) - attach to IRE to keep other proteins away - block translation
5'-IRE if IRP bound to IRE before the coding region -> block translation / affect translation
3'-IRE if IRP boudn to IRE located behind the coding region -> prevent endonuclease from chopping it off(affect mRNA stability)
if high iron level, IRP-1 is bound by 4Fe-4S cluster and becomes aconitase, while IRP-2 is ubiquinated and degraded. -> ribosome can bind and translate the coding region
5'-IRE regulate production of ferritin, at low iron, ferritin expression is blocked
3'-IRE regulate transferrin receptor and DMT1 expression
- at low iron, TFR and DMT1 are needed so IRP is bound to 3'-IRE and prevents mRNA from endonuclease
- at high iron, TFR and DMT1 are no longer needed so IRP-2 is degraded, and mRNA is cut by endonuclease
hemochromatosis - bronze diabetes
- deposition
liver - cirrhosis, HCC
heart - cardiomyopathy
pancreas - diabetes
pituitary - dysfunction
Intestinal iron absorption controlled by regulation of iron transfer from epithelial cell to circulation
Iron cannot be excreted through liver or kidneys
- hepcidin controls iron homeostasis
- short lived, cleared from kidneys, detectable in urine
- binds to ferroportin, moves it to lysosome to have it degraded
- affects ferroportin on intestinal cells as well as macrophages
Three mutations that cause hemochromatosis
- mutation on hepcidin gene (direct)
- mutation on ferroportin (no longer responsive to hepcidin)
- mutation on HFE, TFR2, HJV - affect hepcidin expression, make it defective (indirect)
Iron-refractory iron deficiency anemia (IRIDA)
mutation on TMPRSS6
normally in iron deficiency, hepcidin should be little or none, ferroportin should be maximally expressed to increase iron load
however, in IRIDA, hepcidin is increased, decreasing iron uptake -> anemia
hepcidin expression
(+) - increased Fe, inflammation
(-) - decreased O2, increased erythropoiesis
LECTURE GENE EXPRESSION - part I
First nucleotide on mRNA 5' has triphosphate, while other nucleotides use up their triphosphate to form phosphodiester bond
Unwinding(downstream of pol) and Re-winding(upstream) activities at both sides of transcription
regulation of transcription initiation
As a machine moves, it continues to have 8-10 DNA-RNA hybrid, upstream DNA-RNA melt, while new DNA-RNA are formed at downstream
Promoter is a cis-acting element- sequence element in the gene that is used to regulate the expression of that gene
Trans-acting element - not part of the gene, usually proteins but are attracted to the gene to affect the gene's expression
Cis-acting elements part of the RNA, not DNA
Initiation - from the point sigma factor binds, up to the point where first nucleotide nucleotide bond made
scaffold complex - temporary structure for holding workers and materials
sigma factor has to leave promoter for polymerase to enter elongation
at elongation, polymerase is tightly bound to the transcript while being able to move, w/o sigma factor
DNA dependent RNA polymerase
type I - makes rRNA
type II - mRNA
type III - tRNA and 5s rRNA
RNA polymerase II, subunit I
catalytic core between subunit 1 and 2
C-terminal domain (CTD)
repeat of YSPTSPS
function - initiation, elongation, capping, splicing, termination
differential phosphorylation of Serine residues on CTD of polymerase
- different CTD states recruit different factors
pre-initiation assembly
- trans-acting elements - transcription initiating factors
- general aspect of all transcription initiating factors are that:
they sit on the DNA, and they make it bend
- multiple proteins, one notable example is TFIID - has protein called TATA-binding protein
- recruit TFIIA, TFIIB, then recruit TFIIE, TFIIH => formation of pre-initiation complex, scaffold complex?
know the names of general transcription factors
TFIIA
TFIIB
TFIID (TBP, TAFs)
mediator
TFIIF
TFIIE
TFIIH
SAGA (TAFs, Spts, Adas, Sgfs, Gen5, Tra1, Ubp8)
promoters
- 4 types of elements: BRE, TATA, INR, DPE
- BRE - TFIIB
- TATA - TBP
- INR - TFIID
- DPE - TFIID
- need at least one to be a promoter
- having more than one will make it a stronger promoter
****
Majority of trans-acting factors read DNA major groove surface to detect its sequence information, in order to bind to specific sequence
- accessible
- provides rich information
- so trans-acting factors put an amino acid side chain into the major groove
- Hydrogen bonds used to detect, since its very linear bond, can be very specific
activators have DNA binding domain(for cis element on the gene), as well as protein-protein binding interaction domain
(e.g. estrogen receptor)
activators work through mediator complex
repressors inhibit activator by:
- competitive DNA binding(if activator binding site and repressor binding site overlap), or
- masking the activation site of activator(via physical interaction with activator), or
- direct interaction with the general transcription factors
LECTURE : GENE EXPRESSION II
in transcription before initiation, RNA polymerase complex is bound to mediator, where mediator interacts with activator of cis-element(off-stream activating sequence) on a distant part of genome
how many nucleotides per turn? 10 (?????)
for steroids (e.g. estrogen receptor)
DNA binding domain where receptor binds is distant from ligand-binding domain
estrogen binding does not change DNA, but it changes estrogen receptor conformation
then the change allows binding of co-activator
then interaction between co-activator and mediator turns on the transcription
tamoxifen a competitive inhibitor of estrogen -> coactivator cannot bind
slide 6, sequence below is peptide, which is N terminus -> C terminus
positive and negative elongation factors (e.g. HIV-1 Tat and TAR recruit P-TEFb)
TFIIH phosophorylate 5' end of CTD, then promoters recruit negative elongation factor
and then recruits factors that counteract those factors; positive factors(P-TEFb) come and phosphorylate negative factors(NELF, DSIF), make them leave, which start elongation
TAR RNA at the 5’ end of newly synthesized mRNA strand forms an elaborate stem loop structure that recruits the small protein Tat. Tat ad TAR RNA together recruit the trans acting elements CycT1 and CDK9 (ie recruits P-TEFb)
The HIV-1 LTR promoter is weird in that it is not good at recruiting P-TEFb, and requires this stemloop TAR RNA to do so
most of our DNA wrapped up in nucleosome(histone+DNA), histones
- may obstruct transcription factor's access to DNA
- whereas traditionally histones were only thought to have structural function,
nowadays modifying histones is thought to change transcriptional capacity of many regions of DNA
- histone code read by specific binding proteins that in turn alter chromatin structure
If you recruit histone acetylase to the promoter region -> activate transcription by unwinding nucleosome
histone deacetylase - deactivate transcription by rewinding nucleosome
5' capping of mRNA
- capping enzyme recruited by RNA pol II and binds to its CTD region for activity
- RNA synthesized by RNA pol I and III (rRNA and tRNA) do not get capped
- snRNA, miRNA made by pol II
- cap structure
5' to 5' linkage, 7-methylguanosine
prevent 5' end from being chewed up by exonuclease
- 5' capping also serves as a recognition site for mRNA by ribosomes
3' end formation
- CPSF binds to RNA downstream of AAUAAA, there's a cleavage site
- Once RNA cleaved, poly-A tail added by poly A-polymerase (PAP)
- poly-A tail sequence bound by poly-A binding protein
- contribute to stability of mRNA and for tRNA recruitment
- every mRNA has poly-A tail, except for cell cycle dependent histones (which have conserved stem loop)
LECTURE: GENE EXPRESSION - PART 3
upstream exon AG -------A--Py---G downstream exon
branch site A
exonic enhancers - found at the exons
intronic enhancer - usually near downstream 5' splice site
spliceosome
snRNP, know their roles
U1 - binds to 5' site(RNA recognizes) and then binds to 3' site; its RNA component complementary binding to splice site
U2 - binds to branch site(A) and forms part of catalytic center
U5
U4
U6 - catalyze splicing
So, once U1 bind to 5' and U2 bind to A, U5, U4, U6 come and bind to form catalytically competent spliceosome.
Mechanism of splicing - this happens alongside transcription
1) OH' group from branch point A nucleophilically attacks phosphodiester bond of 5' exon, leaving exon-1-OH floating while forming a lariat via phosphodiester bond with sequence next to 5' exon
2) exon1-OH attacks 3' exon's (exon 2) phosphodiester bond, joining with exon 2 while leaving intron site with OH. => exon1-P-exon2 => a continuous sequence
splicing of intron from RNA requires formation of 3-5 and 2-5 linkages
alternative splicing, look at a,b,c,d,e
- some introns become exons
average size of introns in the human genome is 100 bp to 2kb long
cis acting factors
- exonic intronic silencer enhancer
trans acting factors
- certain types of proteins expressed in certain cells, so alternative splicing can be conducted
splice sites are far away from consensus
exon enhancer or silencer within exon itself
intron enhancer or silencer within 200 nucleotides of the splice site
many of these are RNA binding proteins that bind to these enhancers or silencers, and they help the spliceosome recognize something as exon or prevent the spliceosome recognize something as exon
trans-acting factors, alternative splicing factors
alternative splicing human disease
- fronto-temporal dementia with parkisonism (FTDP-17)
- inclusion or exclusion of exon associated with multiple sclerosis
RNA interference
- stop the translation of mRNA
- downstream of transcription
- done by miRNA/siRNA
- siRNA (double strand) bind to mRNA and cause the strand to be cleaved by spliceosome
- miRNA (single strand that forms stem loop), binds to it and inhibits translation, which is eventually degraded
- miRNA does bind to 3' UTR
- miRNA do not perfecty complementarily bind to celular target
- siRNA perfect complemntarity to the target
- miRNA form stem-loop
gene splicing by double strand RNA
migration pattern (alkaline electrophoresis) vs migration pattern (acid electrophoresis)
