Final exam Flashcards
(246 cards)
1
Q
Signal hypothesis
A
secreted proteins contain a signal telling them where to go
2
Q
How to test the signal hypothesis
A
- Choose a culture a cell type which secretes proteins
- fractionate to isolate ER
- create a cell free system for in vitro translation
- label proteins with pulse chase
- purify an immunoglobulin and translate
- disrupt membrane at different time points and examine size
3
Q
Read out experiment
A
- adding detergent to remove microsome
- when microsomes were disrupted when proteins are still translated
- longer base pairs found
4
Q
Major protein sorting pathways in eukaryotes
A
- cystolic proteins translated in cytosol but some will need to be targeted somewhere due to targeting sequence
- proteins translated on ribosomes attached to RER but then translocated to lumen
5
Q
Experimental evidence for signal hypothesis
A
- direct relationship between a large precursor protein and small mature proteins
- mature secretory protein only produced if microsomes were present
- mature secretory proteins were protected from digestion inside microsomes
- conserved sequence
- found at N terminus which has 6-12 hydrophobic AA
6
Q
Co-translational translocation process
A
- signal recognition particles (SRP) binds to signal sequence to halt translation
- SRP binds to SRP receptor on ER membrane via GTP hydrolysis
- polypeptide transferred into Sec 61 translocon
- polypeptide chain elongates and translocates through channel
- signal sequence cleaved
- polypeptide chain in lumen folds
- ribosomes dissociates and channel closes
7
Q
Sec 61 translocon
A
- conserved protein
- structure has a pore with a plug to form a channel
- chain elongation at ribosome is sufficient to drive polypeptide through channel
8
Q
Integral membrane protein path
A
- ER
- Golgi
- Membrane
- Lysosome
9
Q
Where are integral proteins synthesised
A
RER
10
Q
Topogenic
A
orientation and number of times a polypeptide crosses the membrane
11
Q
Type 1 integral protein
A
COO- in cytosol and NH3 signal in ER
12
Q
Type 2 integral protein
A
NH3 in cytosol and COO- in ER
13
Q
Type 4 integral protein
A
have loops with NH3 in ER and COO- in cytosol
14
Q
Getting proteins into the membrane
A
- new polypeptide chain-ribosome complex associates with translocon
- signal sequence cleaved and translocated
- stop transfer anchor sequence of hydrophobic AA is translated and enters the translocon
- no more translocation into lumen
- stop transfer anchor moves laterally into membrane via translocon cleft
- polypeptide anchor
- translocon closes
- ribosome dissociates
15
Q
Mitochondria
A
- contains own DNA but cannot make all their own proteins
- grow and divide via the uptake of cellular proteins and lipids
- pre proteins for matrix have amphipathic alpha helix signal sequence
16
Q
Post translational translocation
A
- pre protein synthesised on cytoplasmic ribosomes are kept unfolded
- pre protein binds to Tom20/22 receptor on the mitochondrial outermembrane
- Tom20/22 transfers pre protein to Tom40 pore
- Tom40 passes pre protein to Tim complex in the inner membrane
- pre protein transferred int matrix
- Hsp70 chaperone binds and uses energy from electrochemical gradient to cleave signal sequence
- protein now active in mitochondrial matrix
17
Q
Methods to study secretory pathways
A
- In vivo = radiolabel AA from secretory tissues
- In vitro = live imaging fluorescent fusion proteins in cells
- Conditional yeast mutants = force mutations in cell and look for phenotypes
18
Q
Vesicle budding and fusion to form membrane carrying proteins
A
- initiated by polymerisation of coat protein complexes
- coat proteins bind to cytoplasmic tails of proteins sticking out of ER membrane
- vesicles pulled out
-cargo recruited to membrane proteins and gather in vesicles - vesicles uncoat in the cytosol exposing membrane proteins
- vesicles move through the cytosol via motor proteins
- vesicle fuse to the targeted membrane by SNARE binding
19
Q
Formation of COPII vesicles from ER
A
- Sar1 GTP protein binds to sec 12 receptor in the membrane
- exchange of GDP for GTP to energise the protein
- Sar1 undergoes a conformational change which makes the N terminus tail to stick into the membrane of ER
- Sar1 binds to Sec23/24 (coat protein components)
- vesicle formation
20
Q
What causes uncoating
A
GTP hydrolysis which allows the vesicle to fuse with the golgi
21
Q
What does having more coat proteins do
A
attracts more proteins with signal
22
Q
How are vesicles moved along microtubule tracks
A
Motors
23
Q
Anterograde motors
A
forward moving
24
Q
Retrograde motors
A
backward moving
25
Movement from ER to golgi
- Anterograde
- CopII vesicle initiated by Sar1
- Dynein motor
26
Movement from golgi to ER
- retrograde
- CopI vesicles initiated by Arf
- Kinesin motor
27
CopI vesicle roles
- initiated by Arf (GTP binding protein)
- recycles back ER proteins
- returns missorted resident ER proteins with retrieval signal
- binds selectively to receptors based on pH
- binds tighter under acidic conditions in the golgi and less then less acidic conditions (ER)
28
RER retention signal for soluble proteins
KDEL
29
ER export signal
Di-acidic sequence which is found in the cytoplasmic domain of membrane cargo proteins
30
ER retentional signal for membrane proteins
Lys-Lys
31
Signal that targets proteins to nucleus
Nuclear localisation
32
Cisternal maturation
when vesicles fuse with the cis golgi they form new cis golgi and move forward and everything gets pushed forward into trans golgi
33
Role of post translational modifications
- quality control (tags misfolded)
- structural stability
- production of distinct molecules for signalling
- activation/inactivation of enzymes
34
4 main post translational modifications
- folding and assembly of multi subunit proteins in the ER
- disulfide bond formation in the ER
- glycosylation modification in ER and golgi
- specific proteolytic cleavages to activate/inactivate occurs in ER, Golgi and vesicles
35
N-linked glycosylation
- occurs in ER
- precursor oligosaccharide (3 different branches of sugars) detects signal sequence and binds to protein
- 3 sugars are removed by glycosidases
- protein is now ready to move to the golgi
- travelling to golgi then more sugars are added
36
glycosylation signal
Asn-X-Ser/Thr
37
Vesicles that bud from trans golgi structure
- inner layer = adaptor proteins
- outer layer = clathrin
38
adaptor proteins
bind cytosolic domains of membrane proteins to determine what cargo is to be transported
39
Dynamin role
polymerises around neck of bud and stretched neck untill it pinches off using GTP
40
Lysosomal sorting signal
mannose-6-phosphate
41
Lysosome
- digestive and recycling compartments to break down waste macromolecules to monomer building blocks
- has digestive enzymes which are active in acidic pH of lysosome
- pH maintained by H+ pumps
42
Where is the lysosome signal added to the protein
cis golgi
43
Protein from golgi to lysosome
- M6P receptor binds to M6P signal
- protein recruited in clathrin/AP1 coated vesicles
- receptors recycled
- vesicle uncoated via Arf
- change to acidic pH = endosome formaton
- dephosphorylation and activation of protein
- endosomes fuse into lysosome
44
Lysosomal storage disorders
- Battens disease = mutation in gene coding for lysosomal enzymes which leads to accumulation of lipids as lysosomes cant form
- No M6P signal
45
Regulated endocytosis
same process as lysosome transport but uses AP2
46
Regulated secretion of proteins from trans golgi
vesicles released due to a signal (insulin)
47
Continuous secretion of proteins from trans golgi
protein is always secreted (albumin), typically has no coat proteins
48
LDL signal sequence
Asn-Pro-X-Tyr
49
Receptor mediated endocytosis of LDL
- LDL binds to LDL receptor
- endocytosis
- AP2 clathrin vesicle formation
- targeted to endosome
- dissociates from receptor due to pH
- receptor recycled
- lysosome formation
- LDL broken down into AA, FA and cholesterol
50
Familial cholersterolemia
- mutation in LDL receptor
- increase in cholesterol
- heart disease
51
LDL receptor
Type 1 transmembrane protein
52
Uniporter
movement of a single molecule down gradient
53
cotransporters
- symporter and antiporter
- couple transport of 2 different molecules
54
pumps
hydrolyse ATP to transport ions against their gradient
55
Similarities between channels and transporters
- made of multiple membrane proteins that assemble in lipid bilayer to form an aqueous pore
- regulated or gated
- can undergo a conformational change
- chemical energy coupled to movement
56
Peter agre
inserted mRNA aquaporin into frog egg and saw cell burst which indicated water movement
57
where are there a high concentration of aquaporins
- kidneys
- intestines
- plant roots
- in desert mammals kidneys to avoid dehydration
58
GLUT 1
- a uniporter that takes glucose into the plasma membrane of cells through facilitated transport
- has 2 conformational states which changes by glucose binding
- limited
59
Vmax
- maximum transport rate
- achieved when concentration gradient is large
- uniporter working at max rate
60
what determines the rate of transport
affinity
61
Km
- affinity of a transporter for its substrate
- concentration of substrate at which transport is half Vmax
62
GLUT 1 Km
- decreased Km
- efficient as high affinity for glucose
63
GLUT 2
- uniporter in pancreas
- high Km allows it to be a glucose sensor
- low affinity for glucose
64
Why is there a low cytosolic concentration of glucose
- rapid phosphorylation of glucose to G6P
- allows constant import of glucose
65
GLUT 4
- in muscle and adipose cells
- stored in vesicles attached to golgi
- insulin responsive
66
insulin and GLUT 4
- insulin binds to receptors on muscle cells
- signalling occurs
- kinesin transports vesicles
- GLUT 4 receptors are inserted into plasma membrane
- increase glucose uptake into cell
- decrease glucose concentration
67
What happens when there is low glucose
- GLUT4 endocytosis
- transport to endosome
- GLUT4 recycling
68
sodium glucose symporter
- transports glucose into cells when the outside concentration decreases
- transports 2 Na down gradient and glucose against gradient
- energy released by Na movement powers transport
- found in intestinal and kidney tubule epithelial cells
69
Types of ion pumps
- P class
- V class
- F class
70
P class pump
- found in plasma membrane
- tetramer
- has catalytic and regulatory subunits
- ionic composition of cytosol kept constant
- Na/K pump and Ca pump
71
V class pump
- found in lysosomes, endosomes and vacuoles
- pump protons due to acidity differences
- keep inside of lysosomes, endosomes and vacuoles acidic
- balanced by facilitated diffusion of Cl to maintain electrical neutrality
72
F class pump
- found in inner mitochondrial membrane
- pump protons from matrix
73
Na/K ATPase
- Na and ATP binds to pump
- phosphorylation of alpha subunit
- conformational change
- 2K bind and 3Na out
- dephosphorylation
- conformational change
- 2K into cell (cytosol)
74
Transcellular transport of glucose into blood
- Na/K ATPase brings 2K in and 3Na out
- K channel opens and K flows out to set up membrane potential
- Glucose broken down from food in intestine and is moved into the cell by a glucose/Na symporter
- GLUT 2 moves glucose from cell to blood
- Aquaporin moves water from lumen to cell via osmosis
75
Acidification of stomach lumen by parietal cells
- H/K ATPase (P-class) moves protons out of cell into stomach lumen, K into cell
- K channel opens to allow K to flow back
- Cl/HCO3 antiporter opens due to increased pH in cytosol, moves HCO3 into blood and Cl into cytosol
- Cl channel opens and moves Cl from cytosol to stomach lumen
- stomach lumen pH decreases as HCl forms
76
Electrical potential
differential distribution of charged ions on each side of the membrane
77
What interactions generate a membrane potential
K/Na ATPase and K channel
78
K channel structure that allows specificity
side chains of amino acids around the pore which only bind and interact with K
79
excitatory cells
- neurones
- muscle
80
Depolarisation
- Na flows into cytosol down its gradients
- Na channels open and voltage change triggers opening of gated Na channels down axon
81
Repolarisation
- +50mV membrane potential
- no more Na inflow
- voltage gated K moved out of cytosol into exterior
- Membrane potential now -70mV
82
what happens if action potentials arent regulated
- toxicitiy in brain
- seizures
- over expression of inhibitory cells
- parkinsons disease
83
Voltage gated Na channels
- Has alpha helices with amino acids that are positively charged
- when closed the alpha helices are attracted to negative of cytosol
- alpha helices move towards negative exterior due to depolarisation which opens the channel
- when +50mV membrane potential reached then channel inactivating segment blocks channel untill -70mV reached (Refractory period)
84
Optogenetics
uses light activated channels to control cell function through manipulating membrane potential
85
Channel rhodopsin 2
- activated by blue light
- Na moves into cytosol
- stimulates AP
- can control movement
- used to study behaviours, parkinsons, epilepsy and PTSD
86
Synapse
point at which 2 neurons or cells meet
87
Neurotransmitters
- chemical messengers
- synthesised from amino acids in cytosol
88
Neurotransmitter release at synapse
- neurotransmitters imported into vesicle using V class pumps and a proton antiporter
- vesicles move towards membrane
- V and T SNARES bind to form a SNARE complex to dock the vesicle at membrane
- Action potential arrivs
- voltage sensitive Ca channels open
- vesicle uses with synaptic cleft via synaptotagmin binding with SNARE
- neurotransmitter exocytosis into synaptic cleft
- Na/neurotransmitter symporter reuptake of neurotransmitter
- clathrin/AP2 recycles vesicles
89
Botulin toxin
- prevents exocytosis of neurotransmitters at the neuromuscular junction as SNARES cannot fuse
- 2 part polypeptide
- binds to motor neurons to prevent Ach release = paralysis
- protease which cleaves V-SNARE on vesicles so they cannot dock
90
cocaine/amphetamine
- bind and inhibit DAT
- increase dopamine in cleft
- decreased reuptake of dopamine
- increased mood
91
Antidepressants
- act on serotonin reuptake symporter
- increases serotonin in synaptic cleft
- increased stimulation
92
Patterning
how cells develop their fate in space and time
93
Cell lineage
progressive determination of cells with restriction in developmental potential and differentiation into specialised cell types
94
How can cells of c.elegans be tracked during division
using non toxic dye which is shared to daughter cells
95
What type of cell division is c.elegans
asymmetrical
96
The orientation of what determines the nematode body
blastomeres
97
what determines the axis of cell division
mitotic spindld
98
What proteins are in the AB
Par 3 and Par 6
99
What proteins are in the P1
Par 1 and Par 2
100
Par protein role
organising the distribution of other molecules and orientation of mitotic spindle
101
Par3 role
inhibits centrosome rotation/rotational development in AB
102
What determines cell-cell interactions
the positioning of cells
103
Cell-cell interactions in c.elegans
- P2 signals APX1 moleule to ABp which induces differentiation via notch signalling
- P2 signals MOM2 to EMS via wnt signalling
- ABa doesnt receive any signals
104
Pop1 accumulation in c.elegans
- Pop1 accumulates in mesoderm
- pop 1 degraded in endoderm due to mom5
- cell becomes polarised
105
How to see if cell to cell signaling is important for development
seperate or remove cells to see the impact on differentiation
106
Drosophila cell division
- nuclear division without cytokinesis = karyokinesis
- generates a syncytium (lots of nuclei within a cytoplasm)
- cellularisation = nuclei migrate to egg surface and membrane forms around each
- asymmetric distribution of mRNA at egg
107
Determining factors in drosophila
- anterior = bicoid which binds to hunchback and turns it on
- posterior = nanos
108
unique features of mammals
- slow cell division
- no maternal factors
- rotational cleavage
- asynchronus cleavage
- cells end up in one of 2 microenvironments to determine fate
109
Key events during early human development
- fertilisation
- cleavage
- compaction
- cavitation
- implantation
110
Morula
- compaction of blastomeres
- increased cell to cell adhesion
- new cavity
111
Compaction in mammals
- cells flattening
- increased cell to cell adhesion
- formation of epithelium
- formation of tight junctions between epithelial ells
- becomes polarised
112
when does differentiation start in mammals
32 cell stage
113
inner cell mass
embryo fate
114
external cells
fated to trophoblast
115
cavitation in mammals
- Na moves from uterine across apical surface of trophoblast
- Na/K ATPase pumps Na from basolateral surface of tm- ophoblast into blastocyst cavity
- increased osmotic pressure
- Cl/HCO3 exchange between trophoblast and blastocyst cavity to maintain electrical neutrality
- water moves into blastocyst
- embryo swells
- cavity forms
116
blastocyst expansion
- cavitation = embryo swelling
- same number of cells and cell volume
- different cell shape
- increased blastocoel volume
117
Epithelial expansion
- radial cleavage via horizontal plane
- increases number of cells
- same cell volume
- increases blastocoel volume
118
Radial cleavage
generation of trophoblast cells with a polarised side
119
Tangential cleavage
- vertical plane
- generation of inner mass cells
- one non polarised
- one polarised
120
What happens if there are too few cells in the morula
all cells will be outside cells and no embryo develops
121
Pluripotent
gives rise to embryonic tissue
122
Totipotent
gives rise to whole embryo and placenta
123
When do embryo cells become totipotent
8 cell stage (seen in mice)
124
Implantation in embryos
- trophoblast cells adhere to uterine epithelium
- secrete proteins and hormones to the endometrium
125
IVF
- blastocysts are uterine transferred
- embryo screening can be done for x linked diseases (cystic fibrosis + downsyndrome)
126
Ectoderm fate
- skin
- nervous system
127
mesoderm fate
- muscle
- skeleton
- blood
- kidney
128
endoderm fate
gut
129
how epithelial cells change shape
using an adhesion cytoskeletal belt which contracts
130
epiboly
- cells flatten and spread
- thinner cell layer
- increased surface area
- increased cell substratum adhesion
131
intercolation
- cells intermix and spread to form a single sheet
- change in cell to cell adhesions
132
convergent extension
- elongation of tissue mass
- changes in cell to cell and cell to substratum adhesions
133
invagination
- localised areas of cell constriction causing buckling and bending of sheet
134
involution
- folding of cell layer after invagination
- extensive
135
migration
- cells move away from edges of coherent mass (Single cells)
- decreased cell to cell adhesion
136
ingression
- cells detach from epithelial layer and migrate into basal extracellular matrix
- changes in adhesions
- single cells drop out
137
proliferation
- cell layer expansion
- can lead to localised growth at an edge or folding and bucking
138
Frog grastulation
- Cleavage
- bottle cells invaginate
- bottle cells movie via involution
- ectoderm spreads via epiboly and intercolates
- mesoderm stretches via convergent extension
- new cavity forms
139
Sea urchin
- invagination of endoderm
- convergent extension of endoderm
- mesenchymal cells extend filopodium and tow archenteron via ingression
- ectoderm cells pulled inside via involution
140
spemann mangold organiser
region where bottle cells are found which signals to neighbouring cells
141
difference between bottle cells and neighbouring cells
- bottle = autonomous/ defined fate
- neighbouring = condition/location dependent
142
nieuwkoop centre
- determines where gastrulation will start
- when sperm enter egg it causes corticol rotation which moves maternal factors
- movement of maternal actors activates wnt signalling
143
Mesenchymal cell features
- secrete ECM
- flattened
- motile
- expresses vimentin
144
epithelial cell features
- polarised cell sheet
- specialised tight junction
- attached to basement membrane
- expresses cytokeratin
145
somite
- form in anterior to posterior direction
- ball of mesodermal tissue found near segmented vertebral column
- staining with e cadherin you can see the new somites due to increase in epithelial cells wheras old one have increase in mesenchymal
146
How to study EMT signals
- genetic models
- in vitro culture systems
147
drosophila genetic model EMT
- twist and snail genes are activated by maternal dorsal
- twist turns on mesenchymal fate (transcriptional regulator)
- snail turns off epithelial fate (transcriptional inhibitor)
148
In vitro EMT system
- grow epithelial cells and add growth factors to induce and EMT event
- observation = down desmosomes, downn adherens junctions, down cytokeratins, up motility, up spreading, up vimentin
149
where do MET events occur
uteric bud and metanephrogenic mesenchyme
150
MET event in kidneys
- metanephrogenic mesenchyme and uteric bud grow together
- MM induces UB to branch
- tip of each branch MM condenses to undergo a MET
- epithelial cells differentiate, proliferate and elongate to start forming nephron
- basement membrane degraded to form a constant tube
151
stem cells
cells that divide to form one daughter that goes on to differentiate and one daughter retains stem cell properties
152
Why we need stem cells
- growth
- renewal
- repair
153
Multipotent
gives rise to all cell types of a specific tissue
154
unipotent
gives rise to one type of cell
155
stem cells giving rise order
- stem cell
- restricted stem cell
- prognitor cell
- terminally differentiated cell
156
Epidermis layers
- basal layer = keratinocyte stem cells reside here to be protected, proliferation occurs
- keratinocytes differentiate and move upwards
- outer layer = keratinocytes flatten and lose their nucleus and die
157
Drosophila stem cells neuroblasts
- neuroblast (with bazooka, pins) drops out of epithelial cells via ingression undergo an EMT event
- bazooka and pins = stem cell proteins
- miranda accumulates
- asymmetric cell division = one stem cell and one mother cell
158
What can embryonic stem cells give rise to
- epithelial cells
- neurons
- embryoid body
159
transgenic mice using embryonic stems experiment
- isolate single cells from blastocyst of black mouse and grow in culture
- clone and grow single cell for many generations to generate embryonic stem cells
- transfect embryonic stem cells with a transgene and antibiotic present marker
- selects cells in presence of antibiotic
- inject cells into blastocysts of white mice
- transfer embryos into surrogate mother
- pups will be a mix of black and white = chimera
- chemira and white = grey mice (heterozygous)
- grey and grey = black mice (homozygous)
160
Transcription factors that keep embryonic stem cells in their pluripotent state
- Oct 4
- Nanog
- Sox2
161
what do the transcription factors do
- activate genes for self renewal and pluripotency
- repress genes that induce specific differentiation pathwars
162
Medical applications with induced pluripotent stem cells
- remove differentiated cell and grow in culture with yamanaka factors to induce stem cells
- can be disease specific drugs or repair disease causing mutations
163
problems with induced pluripotent stem cells
- no consensus yet on most consistent or optimal protocol
- check for culture induced changes aka activation of oncogenes
- difference with methylated regions in IPS and ES so they have to be treated differently
164
Components of plasma
- blood protein, water and RBC
165
Immunity
the ability to defend against infection and distinguish between self and altered self
166
Antigen
a molecule that elicit an immune response
167
Features of an immune system
- specificity
- diversity
- memory
- tolerance
168
Types of immunity
- mechanical and chemical defences
- innate
- adaptive
169
Types of cytokines
- chemokines
- interleukins = differentiation and function of T + B cells
- interferons = antiviral
- tumour necrosis factors = cell death
170
What can flow cytometry measure
- fluorescently labelled antibodies
- size
- complexity
- proteins secreted by a cell
171
Flow cytometry method
- cells are isolated
- antibiotics added
- stain for surface or intracellular markers
- run sample
- cells seperated by laser
- graph plotted
172
Primary lymphoid tissue
where cells are generated (T cells = thymus, B cells = bone marrow)
173
secondary lymphoid tissue
site where adaptive immune responses are generated
174
Stromal cells
provide structural support, organise discrete zones and secrete soluble mediators for cell survival
175
Innate immunity
first line of defence = physical and chemical
176
skin epidermis
- produce a permeable barrier
- multiple layers of keratinised epithelial cells
- has a sebaceous gland to produce oils to prevent pathogens
- sheds outer layers to remove pathogens
177
antimicrobial peptides
- defence peptides that are amphipathic with cysteine residues
- secreted by phagocytes and epithelial cells
- disrupts bacterial membrane by causing rupturing (Defensin)
178
What is the complement system
- 30 proteins travelling in the blood which are activated by infection
- proteolytic cascade
179
complement system pathways
- classical = activated by antibodies
- lectin = activated by lectin
- alternative = activated by parts of pathogen
- ALL ACTIVATE C3
180
C3 activation results in
- C3a and C5a chemoattractants activate and recruit immune cells
- C3 binds to other complement proteins to form the MAC creating pores to kill cells
- optimisation of pathogens = macrophage
181
Different types of phagocytes
- dendritic cell
- macrophage
- neutrophil
182
dendritic cell
take up antigens and present on surface to lymphocytes
183
macrophage
phagocytose bacteria and present antigens
184
neutrophil
ingest pathogen and release enzymes to kill them
185
what do phagocytes recognise
PAMPS using Pathogen recognition receptors
186
Pathogen recognition receptors (PRR)
- toll like receptors found at cell surface to recognise external components
- can be inside for internal components
- binds to PAMPS to allow for production of cytokines and pahgocytosis
187
Natural killer cell activation
- controlled by signalling between activating and inhibitory receptors
- inhibitory = if normal cells are present, recognises MHC class 1
- activating = if infected cells
188
Signs of inflammation
- redness and swelling due to vasodilation
- pain
- heat
189
function of inflammation
eliminate cause of infection and create an environment for adaptive immune response
190
inflammation response
- bacterium expresses PAMPS which are recognised by dendritic cells
- dendritic cells secrete cytokines and present antigens
- natural killer cells kill infected cells
- neutrophills lyse bacteria and release reactive oxygen species and phagocytose
- antigen moves to lymph node for adaptive response
- mast cells activated and secrete histamine which increased vascular permeability = swelling = vasodilation
191
B cells
recognise intact antigens in lymph, blood plasma and interstitial fluid
192
T cells
recognise antigenic fragments that are processed and presented by MHC molecules
193
MHC class 1 and 2 similarities
- encoded by HLA genes
- bind peptides to present to the t cells
- membrane proteins
- peptide binding stabilises structure
- lots of sequence variation = highly polymorphic
194
MHC class 1 characteristics
- single alpha polypeptide which spans membrane
- has a beta microglobulin which doesnt span the membrane
- epitope binds to alpha polypeptide
- expressed in all nucleated cells
- activates CTL/CD8 cells
- endogenous antigens
- peptides are 8-11AA long
- coded for by 3 genes
195
MHC class 2 characteristics
- 2 identical polypeptide chains that both span the membrane
- epitope binding side is both alpha and beta peptides
- expressed by antigen presenting cells (dendritic, macrophages, B cells)
- activates helper t cells/CD4
- exogenous proteins
- peptides are 13+AA long
- encoded by 3 genes
196
MHC restriction
complementarity between the peptide and MHC and also between the T cell receptor and MHC
197
peptide loading for MHC class 1
- dysfunctional proteins targeted for proteolysis with u6 addition signal
- protein moves to proteosome
- immunoproteosome cuts protein into specific lengths for MHC
- peptides move into RER using TAP with ATP binding casette
- peptides bind to MHC class 1
- chaperones dissociate
- MHC moves to cell membrane
198
virus strategies to interfere with MHC class 1 immune detection
- MHC not presented
- unfunctional proteasome prevents protein being cut into peptides for MHC
- adenovirus competes with TAP to prevent docking of MHC
199
MHC class 2 antigen presentation
- exogenous protein taken up
- protein enters lysosome to be broken down
- MHC class 2 assembled in ER
- MHC class 2 transported to endosomes with invariant chain via golgi
- invariant chain is cleaved off in endosome leaving a CLIP fragment
- CLIP blocks binding of peptides to MHC
- HLA-DM binds to MHC to release CLIP
- peptides bind to MHC
- MHC travels to cell surface
200
cross presentation
where exogenous antigens can be loaded into MHC class 1 - occurs mostly in dendritic cells
201
characteristics of adaptive immune system
- specificity
- memory
- tolerance
202
cellular immunity
cytotoxic t cells and helper t cells which are directed against intracellular pathogens
203
humoral/antibody mediated immunity
b cells which are directed against extracellular pathogens
204
clonal deletion
removal of self reactive lymphocytes
205
clonal selection
a single lymphocyte gives rise to many lymphocytes that express the same antigen specific receptor
206
clonal selection hypothesis
- each lymphocyte expresses an antigen specific receptor
- binding of antigen to receptor results in lymphocyte activation
- activated lymphocyte will differentiate to produce effector cells with the same receptor
- lymphocytes with self antigen receptor will undergo clonal deletion
207
BCR vs TCR
- BCR = light and heavy chain
- TCR = alpha and beta chains
208
t cell activation primary response
- t cell enters lymph node via blood
- t cell scans dendritic cell for cognate antigen on MHC
- t cell is activated and induces proliferation and differentiation to effector and memory cells
209
TCR complex
- made of TCR, CD3 and CD4/8 coreceptors
- bind to distinct MHC which amplifies signal from CD3
- keeps proteins docked
- induces signalling
210
3 signals required from dendritic cell to activate CD4 T cell
- signal 1 = signal from CD3 in TCR complex
- signal 2 = co stimulatory molecules expressed by dendritic cells which promotes survival and proliferation
- signal 3 = cytokines
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T cell secondary activation/memory response
- binding of TCR to its cognate receptor-MHC complex is enough to induce response
- allows CTL to respond to infected cells directly
- CD4 activates B cells
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diminishing t cell activation
-upregulation of CTLA4 which inhibits function of T cell
- functions as immune checkpoint
- tumour cells upregulate PDL1 which binds to PD1 on T cell to prevent its action
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CTL/CD8+
- kill infected cells expressing MHC1 loaded with their cognate antigen at the cell surface
- done with high specificity
- releases granzyme and perforin to induce death
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types of helper T cells
- TH1 = drive cellular immunity and promotes macrophage function
- TH2 = promote antibody production
- TfH = makes antibodies
- TH17 = involved in inflammation
- Treg = inhibit immune responses
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discovery of antibodies
- serum from guinea pigs injected with a sublethal dose of diphtheria toxin protects other guinea pigs that are exposed to a lethal dose
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passive immunity
immediate response that transfers antibodies from someone who has recovered from the disease, short term immunity
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active immunity
delayed and long term response when exposure to a disease results in an immune response that leads to antibody production
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Antibody isotypes
- IgM = 1st antibody produced
- IgA = in bloodstream and mucosal
- IgE = for parasites and allergy
- IgG = neutralises viruses
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polyclonal
response mediated by many B cells responding to different parts of one virus
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B cell primary response
- activation requires more than 1 signal
- dendritic cell acquires antigen and moves to lymph node
- DC presents antigen to CD4/T helper
- T helper differentiates and proliferates to be activated
- T helper interacts with B cell to induce differentiation
- Some B cells secrete IgM
- Rest of B cells secrete other isotypes
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Germinal centres
specialised structure in the secondary follicle of the lymph node
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Germinal centres role
affinity maturation due to somatic hypermutation = selected B cells with highest affinity for cognate antigen
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Role of antibodies
- neutralisation = prevents bacterial adherence
- opsonisation = promotes phagocytosis
- complement activation to increase phagocytosis and lyse bacteria
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ELISA indirect method
- antigen attaches to the well via charge interactions
- well blocked to prevent non specific binding to well
- antibody added
- secondary antibody added with conjugate enzyme
- substrate added
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secondary immune response antibody isotype presence
IgG >> IgM as it has a higher affinity due to affinity maturation
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Unimmunised cells
- decreased frequency of B cells
- IgM
- low antibody affinity
- low somatic hypermutation
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Immunised cells
- high frequency of B cells
- IgG and IgA
- high antibody affinity
- high somatic hypermutation
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Types of vaccines
- Live attenuated vaccine (live pathogen which is less virulent)
- Inactivated pathogen
- protein subunit and VLPS (protein component of pathogen which lacks genetic material)
- toxoid
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Adjuvants
stimulate immune response by activating innate receptors which increase co-stimulatory molecules
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Cancer characteristics
- uncontrolled proliferation (autonomous growth signals and lack of growth inhibition)
- escape from apoptosis
- lack of senescence
- angiogenesis (forms blood vessels)
- metastasis and invasion
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How cancers arise
- mutations
- chromosomal rearrangements
- chromosomal losses
- epigentic changes
- viral genes
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What causes changes in DNA
- copying errors
- chromosomal seperation errors
- inheritance
- viruses
- mutagens
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Evidence for a single cell starting cancer
- all tumours from a patient were DNA sequences
- All cells detected a similar mutant genome from a single parent but some continued to mutate (clonal diversity)
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How proto-oncogenes can become oncogenic
- overexpression of normal protein (gene duplication or overactive promoter)
- point mutation
- viruses have homologue copy which increases viral replication
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Signalling molecule oncogene = PDGF-B
- normal platelets have a dimer of PDGF-A and PDGF-B bound to PDGF-R
- V-sis has a homologue of PDGF-B that can still bind to the same sites
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Signal receptor oncogene = EGF-R
- HER1 + HER2
- signaling triggered by dimerisation of receptor when ligand is bound
- HER1 oncogene = deletion of extracellular domain which brings membrane bound areas together so continuous signalling in absence of ligans
- HER2 oncogene = mutation in transmembrane domain which brings receptors together = continuous signalling
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Intracellular transducers oncogene = RAS
- normally is activated by GEF and inactivated by GAP
- oncogene blocks action of GAP so it remains active to increase signalling and proliferation
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Ways to cause loss or mutation of tumour suppressor gene
- mutation resulting in a frame shift
- loss of chromosomes
- loss of heterozygosity (missegregation or mitotic recombination)
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Retinoblastoma
- familial or sporadic
- involves loss of Rb = continuous signalling with E2F
- familial group suggests inherited genetics
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Rb normal role
- in early G1 Rb is bound to E2F
- mid G1 Rb is phosphorylated
- E2F released
- Rb continues to be phosphorylated to prevent E2F binding
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p53 normal action
- blocks cell division by inducing p21 which blocks CDK2
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p53 in cancer and mutations
- cells will carry on dividing even in the presence of damaged DNA
- HPV produces E6 which binds to p53 to ubiquitate it and degrade it
- only one mutant p53 can disrupt its tetramer function = dominant negative
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metastatic disease can
- interfere with normal physiology
- take up too much space
- starve you
244
how to treat advanced cancer
- surgery
- chemotherapy
- radiotherapy
- immunotherapy
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cytotoxic t lymphocytes with cancer cells
can mistake cancer cells for virus infected cells due to the altered protein sequences so they will release granules with perforin and granzymes to kill them
246
immune checkpoints
- stops CTL to protect tissues from excessive killing
- PD1 is common checkpoint
- most immunotherapies block PD-1 pathway to increase the activity of CTL
