Block 3 - environmental perception Flashcards
1
Q
why is environmental perception particularly important in plants
A
because they cant move
2
Q
describe autocrine signalling
A
cells detect a signal they produce e.g. metabolite
3
Q
describe juxtacrine signalling
A
where adjacent cells initiate responses by direct contact
4
Q
describe paracrine signalling
A
cells detect local signal from neighbouring cell e.g. neurotransmitters
5
Q
describe endocrine signalling
A
cells detect a signal secreted by distant cells e.g. hormones
6
Q
what connects stimuli reception to responses
A
signal transduction
7
Q
what is a stimuli
A
something that initiates a response through signalling e.g. physical, adjacent cells, external chemicals, internal metabolites or hormones, small molecules, peptides, gases etc
8
Q
why do we need a range of stimulus receptors
A
because there is a range of stimuli
9
Q
give examples of some responses to stimuli
A
physical movement
physiological and behavioural changes
differential gene expression
10
Q
some receptors require a cofactor, give an example
A
chromophore for light detection - the cofactor detects the light stimulus
11
Q
some receptors associate with the PM in order to regulate ………… ……………. activity which can change ion concentration and activate downstream proteins
A
ion channel
12
Q
receptors activation can stimulate enzymes associated with the receptor. in what 2 ways can a kinase for example be associated with a receptor
A
it can either be an intrinsic kinase or an independent kinase that is recruited to the receptor
13
Q
signalling often amplifies the response level relative to the ………. ……………
A
stimulus magnitude
14
Q
describe protein kinase cascades
A
phosphorylate numerous target proteins and amplify the response. massive amplification
15
Q
what is desensitization
A
it enables cells to avoid excessive responses. receptor activation may lead to negative feedback that switches off the receptor or removes it from the cell. a signalling component feeds back onto the receptor
16
Q
why would continual response to a stimulus be a problem
A
it can damage the cell and it uses valuable resources
17
Q
give an examples of how responses to stimuli are prioritised
A
priority may be given to pathogen stimulus
there is crosstalk between signalling pathways
18
Q
the activity of one receptor can change the activity of another …………..
A
receptor
19
Q
give some outcomes of defects in reception or signalling
A
impair metabolism or development
cause disease e.g. cancer, diabetes, endocrine diseases
20
Q
how does reception and signalling have application in drug design
A
in making inhibitors/activators and modifying pathways and in synthetic biology e.g. optogenetics
21
Q
give 2 examples of a receptor where stimulation activates ion channels that give rise to a response
A
channelrohodopsin activated by light or the nicotinic acetylcholine receptor involved in neural transmission
22
Q
describe channelrohodopsin main function
A
it is a light activated ion channel that functions in phototaxis of unicellular green algae e.g. Chlamydomonas
23
Q
describe channelrhodopsin structure
A
it has one subunit and is a 7 TM protein (7+M spanning helices) that forms an ion channel in the PM. it is the photoreceptor and the ion channel
24
Q
how do Chlamydomonas respond to a light stimulus
A
they move towards it to increase photosynthesis
25
how many channelrohodopsins does Chlamydomonas have
it has 2 - they are both non specific cation channels and have slightly different ion specificities
26
what is channelrhodopsin gated by
light
27
what s the channelrohdopsin chromophore (cofactor that detects light) called
retinal
28
what happens when retinal of channelrhodopsin is excited by light
it generates a conformational change in the channel due to isomerisation of retinal resulting in an influx of ions
retinal changes from all trans to 13 cis retinal
29
what is the maximal absorption for retinal in channelrhodopsin
480nm
30
after opening of channelrhodospsin the channel quickly closes, what causes this
retinal returns to its all trans form
31
channelrhodopsin is similar to rhodopsin used in visual photoperception but what is a key difference between them
rhodopsin isn't an ion channel
32
describe the structure of the nicotinic acetylcholine receptor
it has 5 subunits - beta, gamma, sigma and 2 alpha that bind acetylcholine
each subunit has 4 membrane spanning helices
the ligand binding sites are in the extracellular face of the subunit
the 5 subunits are arranged around a central channel
33
what is the ligand for the nicotinic acetylcholine receptor
acetylcholine or nicotine
34
what happens when the ligand binds to the nicotinic acetylcholine receptor
there is a conformational change that opens the channel to Na and Ca
35
what happens to the nicotinic acetylcholine receptor experiences prolonged ligand exposure
there is a conformational change that prevents ligand binding and channel opening - desensitisation
36
what do neurotoxins do to the nicotinic acetylcholine receptor
they cause receptor inactivation and hence paralysis
37
give 3 examples of receptors that have enzymatic activity (protein kinase activity that is activated on receptor activation)
- bacterial-2-component histidine kinase
- mammalian growth factor TGF beta signalling and its ser/thr kinase receptor
- tyrosine kinase signalling - insulin receptor
38
how do bacteria move toward or away from a stimulus
they use their flagella
39
what are 2 component systems
they comprise a receptor histidine kinase that detects the stimulus and a response regulator that initiates the response
40
describe the receptor histidine kinase of the bacterial 2 component system
it is located on the cell membrane and has a ligand binding domain on the external surface which binds e.g. sugars or amino acids
41
what happens to the bacterial 2 component system when the ligand binds
the kinase activity is activated in an internal domain of the receptor causing autophosphorylation of a histidine amino acid. the receptor histidine kinase relays the phosphoryl group onto an aspartate amino acid of the second component, a cytosolic protein called the response regulator which then initiates the response e.g. regulates flagella movement or gene expression
42
why does phosphatase need to dephosphorylate the response regulator of the bacterial 2 component system
so that it is ready to activate again in response to a new stimulus
43
2 component systems are common in prokaryotes and less common in yeast. what is the exception to this
plants and yeast
44
what is TGF beta
a type of cytokine. it is part of a super family of related proteins with diverse and important functions including regulating growth, regulating cell division, immunosuppression, cell differentiation, dorsal/ventral specification etc
45
how many TGF beta isotypes are there in animals
3
46
by what type of signalling does TGF beta act
through paracrine signalling
47
describe TGF beta synthesis and secretion
1. protein synthesised with a signal peptide and the N terminus which targets it to the ER, and is removed by proteolytic cleavage during translation and translocation into the ER
2. the protein is secreted as a proprotein - the propeptide domain blocks ligand activity (ligand is never released in its active form inside the cell)
3. the propepetide is removed by proteolytic cleavage outside the cell releaseing mature TGF beta ligand
4. the mature ligand is able to initiate signalling by binding to adjacent cells expressing a TGF beta receptor on their surface
48
describe the TGF beta receptor structure
it is a TM protein with ser/thr kinase
it is a heterotetramer - made from 2x type 1 and type 2 receptor
type 1 has ser/thr kinase activity
type 1 molecules become phosphorylated by type 2
the receptor can bind 2 TGF beta molecules
49
what happens to the TGF beta receptor when TGF beta binds
binding of extracellular dimeric TGF beta ligand to a type II receptor causes it to bind to and phosphorylate a type I receptor, activating it
50
what are the detailed steps in TGF beta signalling
1. TGF beat binding causes type II receptor to phosphorylate type I
2. phosphorylated TGF beta type I receptor recruits and phosphorylates SMAD2 or SAMD3 proteins which are signalling proteins and TFs for TGF beta signalling
3. phosphorylated SMAD2 or 3 dissociates from the receptor and binds to SMAD4. get heterodimer of SMAD4 with SMAD 2 or 3
4. the SMAD2/3 - SMAD4 complex activates transcription of target genes in the nucleus. it translocates to the nucleus and recruits other proteins that together promote transcription
51
why are TGF beta recpetor mutations common in cancer
many tumours contain inactivating mutations in TGF beta receptors or SMAD proteins making them resistant to TGF beat growth inhibition
52
name a signalling pathway that recruits a kinase that then initiates a signalling pathway
JAK/STAT signalling
53
what is the role of JAK/STAT signalling
it is involved in the regulation of transcription by regulatory cytokines e.g. RBC formation stimulation by cytokine EPO
54
when and where is EPO secreted
it is secreted by the kidney in response to hypoxia in the cellular environment
55
what happens when EPO binds to its PM receptor
it causes the receptor to dimerise. the receptor then binds the soluble protein kinase JAK. JAK is activated and phosphorylates tyrosine amino acids on the cytoplasmic domain of the receptor. STAT TFs bind to the p-tyr residues of the receptor via the SH2 domain (part of STAT). STAT is now positioned to be phosphorylated on tyrosine amino acids by JAK. phosphorylated STAT is released from the receptor, forms dimers and translocates to the nucleus where it binds to regulatory sequences to stimulate transcription of genes required for RBC maturation
56
give an examples where ligand/receptor binding activates transcription
steroid hormone signalling e.g. estradiol
57
what is estradiol
it is an estrogen steroid hormone that regulates reproductive cycle and development. it is mainly produced in the follicles of the ovaries. estradiol levels change during the menstrual cycle and get lower after menopause. the levels peak prior to ovulation
58
what is estradiol synthesised from
it is synthesised from cholesterol
59
why is estradiol carried in serum bound to a carrier protein e.g. serum albumin
it is relatively insoluble due to being a lipophilic molecule.
60
estradiol can pass through the membrane due to being small and …………. ………..
lipid soluble
61
describe the estradiol receptor and its role
it is an intracellular estrogen receptor which binds estradiol in the cytosol then dimerises and is translocated to the nucleus, activating as a TF
62
describe the estradiol receptor structure and what happens to the structure when it binds estradiol
there are 2 estrogen receptor proteins, alpha and beta, and each binds a molecule of estradiol. the receptor can be a homodimer or a heterodimer and they have different effects on transcription
the dimer/estradiol complex is specific to a DNA sequence, the estrogen response element, to initiate transcription.
63
what happens when estradiol binds to the estrogen receptor
estradiol binding --> receptor conformation change --> association with cofactor proteins and binding to regulatory sequences of target genes
64
what are many of the genes regulated by estradiol involved in
cell growth and division
65
why do different cell types respond differently to estradiol
because cells differ in the type of receptor and level of expression. the coactivators required can also be differently expressed
66
why is the estradiol signalling pathway relevant to breast cancer
estrogen regulates breast cell proliferation. many tumours have increased estrogen receptor expression and proliferate rapidly in the presence of estradiol. tamoxifen blocks estrogen receptors
67
how does tamoxifen work
it is a competitive inhibitor of estradiol binding. it causes an allosteric change which makes the receptor non functional. helix 12 conformation changes such that the receptor becomes inactivated
68
give 2 examples where a receptor functions in conjunction with a G protein (GPCR)
adenylate cyclase/cAMP signalling
| vision
69
give an example where direct interaction between adjacent cells via surface components initiates signalling
delta/notch signalling
| integrins
70
do tidal and circadian rhythms persist in constant lab conditions and explain
yes - they are driven endogenous clock mechanisms, not external environment changes. the rhythms are thought to be Darwinian fitness benefits
71
annual rhythms result from interplay between the …………… …………. and ….. …… that affect reproductive hormones
circadian clock
| day length
72
what are some circadian rhythm properties
persist in constant conditions, period - 24h, phase can be reset by zeitgebers (light, temperature) which ensures synchronisation with the solar day, show temperature compensation (not affected by temperature)
73
clocks are found in all eukaryotes/prokaryotes and some eukaryotes/prokaryotes
all eukaryotes
| some prokaryotes
74
what is entrainment
the process by which an oscillator is synchronised to an environmental rhythm such as light/dark or warm/cold cycles
75
give examples of things that can be used to monitor rhythms
wheel running
| promoter-reporter fusion
76
what methods can we use to identify clock components
mutagenesis - isolation of mutants with altered circadian period etc
cloning of relevant genes
77
what is an actogram
a plot of mouse activity
78
describe the process of promoter-reporter fusions
promoter - DNA sequence upstream of the genes that controls the gene's expression
- fuse promoter to reporter (e.g. luciferase) then introduce to cell or organism. luciferase protein is synthesised rhythmically and emits flashes of light
79
using promoter-reporter fusions what has been identified about the perception of time in Arabidopsis
perception of time is transferred from mother to daughter cells
80
describe the central oscillator principle: negative feedback with a delay
- component inhibits its own formation after a delay to get a rhythm
- component must turn over rapidly so that it disappears by the end of the oscillation
- there must be machinery to kickstart formations of the component again after it has disappeared
81
anything involved in a circadian rhythm needs to have a half life of less than a day - why
so that it can be formed and disappear in one day
82
describe gene expression negative feedback loops
protein A inhibits its own synthesis by inhibiting a positive TF. rate of destruction of protein A and its mRNA must be fast
the time to get transcription regulation represents the delay
83
which gene has been found to be one of the most important in controlling the circadian rhythm
period gene
84
how have most circadian clock genes been identified
using sequence similarities with drosophila genes
85
what is the role of the per and cry genes
they produce per and cry proteins. per and cry mRNA oscillates and so do the proteins, they are all short lived. the per/cry complex forms in the cytosol and moves into the nucleus after a delay o inhibit expression of per and cry genes
86
what does the clk/bmal1 complex do
it can restart expression of per and cry. it is a TF that binds to the Ebox and E'box which are found in the promoters of per and cry and other genes. binding at these sites promotes transcription by changing chromatin structure
87
how does ckl1 control per
it controls accumulation of cytosolic per during the day by phosphorylating it . this leads to destruction of per (marked by ubiquitin and destroyed by the proteasome)
88
name 5 circadian clock genes and their corresponding proteins
```
period - per
clock - clk
cryptochrome - cry
BMAL1 - bmal1
CK1E - ck1
```
89
describe the mouse circadian clock in a flow chart
dawn --> per/cry complex in cytosol and mRNA levels peak --> per/cry entering nucleus and mRNAs falling --> per/cry all in nucleus - mRNAs absent --> dusk --> per/cry being destroyed --> per/cry low --> per, cry mRNA synthesis started by CLK/BMAL1
90
what is CLK
it is a histone acetyl transferase that acetylates histones in nucleosomes
91
what happens when the per/cry complex interacts with the ckl/bmal complex
it blocks transcription by recruiting a histone deacetylase
92
why are many of the clock controlled genes transcription activated by clk/bmal1
because they have E or E' boxes in their promoters
93
mammals have a master clock - what is this and what is the evidence
it is located in the brain suprachiasmatic nucleus (SCN)
| SCN transplant changes the period to that of the donor in mice
94
major organs have the same clock genes, usually running in the same ………….
phase
95
describe how some of the clock outputs are particular to specific tissues
heart - diurnal variations in cardiac electrical properties and metabolism
skin - skin cells boost DNA repair and do DNA synthesis of growth cycle at night
96
how can rhythms in the liver be reset
by feeding
97
what can circadian misalignment cause
many issues including metabolic diseases
98
how can light interrupt the phase in light sensitive animals
for animals in a light dark cycle, a pulse of light in the early night results in a phase delay. a light pulse in the late night gives a phase advance
99
how is the eye connected to the SCN
by the retinohypothalmic tract
100
light activates the expression of ……. genes. the photoreceptor (…………….) absorbs light in intrinsically photosensitive retinal …………... cells and sends signal to the SCN which leads to creb ………… and clock regulation
per
melanopsin
ganglion
phosphorylation
101
why are blue light filters important
exposure to blue light in the evening will increase per expression and delay sleep (phase delay). exposure later in the night enhances the rise (phase advance
102
the clock and day length control flowering by the level of ..………… protein
constance
103
describe 4 plant activities which are controlled by the circadian clock
- leaf movements - optimizes light capture - low in the morning/night and raised in the middle of the day
- stomatal opening and closing - allows gas exchange during the day but not at night generally
- expression of genes encoding light harvesting proteins - need to be expressed late at night every day
- hypocotyl extension
104
name 2 circadian associated processes in plants
photoperiodic flowering in plants and tuberisation in potatoes
105
what is the Linnaeus flower clock
schematic showing that different plants open at different times of the day
106
what are the key genes/proteins in the Arabidopsis circadian clock
- CCA1 and LHY are a both dawn expressed TFs that bind to the evening element which can inhibit expression of some genes and activate others
- pseudo response regulators - DNA binding proteins expressed between morning and evening
- the evening complex (ELF3, ELF4 and LUX) expressed at dusk and early night
- GI (gigantean) - drives expression of CO which is an output gene that controls flowering
- CRY1 and CRY2 (cryptochromes) absorb blue light - they are not part of the clock itself but are part of the input pathway by which the light absorbed affects the clock
- a gene expressed in the evening or at night is repressed by CCA1 and LHY during the day
107
the Arabidopsis clock is more complex than the mammalian clock but has the same principle which is
feedback with delay
108
what does the fact that the Arabidopsis and mammalian clock components are different tell us
that they evolved separately
109
describe how the plant clock is involved in flowering
LHY and CCA1 go up and inhibit EC expression, the EC therefore goes down so PRR expression goes up. PRRs inhibit CCA1 and LHY so we get overall negative feedback (look at triangle diagram in notes)
110
what 3 types of plants do we get in terms of flowering time
long day, short day and day neutral
111
chyrsantheums and rice are long/short day plants
short
112
under long days, chrysanthemums grow ………. then they are grown in short days just before they are needed and they will ……………..
vegetatively
| blossom
113
in flowering, ……… protein is produced in leaves in response to CO and acts at the ………. ………. …………...
FT
| apical shoot meristem
114
list some factors that affect flowering time
```
clock
temperature
environmental factors
sugars
endogenous factors
GA etc
```
115
is Arabidopsis a long day or short day plant
long day
116
plants sense the length of the light/dark period using the circadian clock
dark
117
what is vernalisation
when some plants need a cold period before they flower
118
what is the GI mutant
a late flowering plant mutant - grows much larger than WT before flowering
119
how is FT expression controlled
it is controlled by the level of CO protein via the circadian clock and also by vernalisation via FLC
120
what is the action of CO in long day plants
- CO is stabilised by light and activates FT expression
- CO is antagonised by FLC, hence FLC inhibits flowering
- exposure to cold reduces expression of FLC and allows flowering
121
what is the action of CO in short day plants
CO inhibits FT production instead of activating it
122
what are the steps in controlling flowering time in Arabidopsis
- the circadian clock drives expression of G1 in the evening/night which drives coexpression --> FT production and flowering
- in a plant that requires vernalisation, FLC levels are high until the plant is exposed to cold. FLC can block the effect of CO on FT expression but vernalisation will reduce FLC expression so flowering is allowed
- sunlight is required by CRY2 to stabilise CO - without CO expression we don't get flowering
(see extended triangle diagram)
123
why do CO and FT only accumulate in long days and what does this mean for flowering
because CO is stabilised by light - if they only accumulate in longs days we will only see flowering in long days
CO rises towards Zt 10 or 12, reaches its peak then falls again
124
at critical night length why don't short day plants flower and why are long day plants different
only get CO message in dark and as a result we get no CO protein because it is unstable in the dark
no FT and no flowering
in long day plants the CO message occurs when it is still light so CO is stabilised and we get FT and flowering
125
describe the sheep annual reproductive rhythm
sheep become fertile in autumn and lambs are born in spring due to the melatonin peak being much longer in short days than long days
long duration of melatonin reduces cAMP which affects the expression of circadian clock genes
per and cry expression overlaps in summer but in winter one is expressed earlier than the other (qualitative and quantitative differences in the transcriptome in winter vs. summer)
the gene for TSH is expressed more in spring/summer than autumn/winter --> seasonal hormone changes --> reproduction control
126
list some human implications of the circadian clock
- shift work - circadian misalignment - associated with increased BMI, T2D, metabolic syndrome (can be rescued with restricted feeding time in mice), stroke, heart attack risk increased
- sleep disorders
- ageing - clock becomes less robust with age
- social jetlag - weekend behaviour patterns
- clock controls DNA repair - shift work has been described as a carcinogen
127
describe the sleep disorder FASPs
familial advanced sleep phase syndrome results from mutations that alter phosphorylation of hPER2 which affects hPER2 turnover rate and can shorten circadian period. in normal diurnal conditions, a short circadian period leads to a phase advance - the clock is reset by light every dawn but gets to the end of the cycle before the next dawn
one with FASPS is active in the first part of the day but sleeps very early and wakes up at 2am - very short circadian period
128
what are the 2 types of mutations that cause FASPS
1. hPER2 - ser662 --> glycine (cant be phosphorylated)
2. CKI gene (controls phosphorylation). thr44 needs phosphorylated to be active but is mutated to alanine which is not phosphorylated -->CKI inactive --> PER not phosphorylated --> FASPS
129
what is the chronotype
mid time of sleep on free days with no social interaction
130
younger people have higher/lower chronotypes than older people
higher - why we don't like 9ams
131
the further east the higher/lower the chronotype
lower
132
how does sildenafil work in jetlag
it accelerates re-entrainment of circadian rhythms after advancing light schedules
133
how does sildenafil work in penile function
it is the same as Viagra. it inhibits cGMP phosphodiesterase and therefore elevates the level of cGMP in target tissues --> smooth muscle relaxation --> increased blood flow --> erection enabled
134
does sildenafil work for a phase delay
no it only works for a phase advance - it elevates cGMP in the SCN allowing re-entrainment
135
CO drives flowering in long/short days plants but prevents it in long/short day plants
long
| short
136
Ca exerts ……… regulatory effects on many enzymes and proteins
allosteric
137
what is the resting Ca concentration in the cytosol and how does this compare to the extracellular concentration
100nm
| much lower than the extracellular concentration
138
what is meant by an allosteric affect
binding --> conformational change --> change in activity
139
name some processes dependent on Ca
muscle contraction
neuron transmission
cell motility
fertilization etc
140
describe the plasma membrane Ca ATPase (PMCA)
ATP dependent pump which actively pumps Ca out of the cell
141
apart from PMCA how else can ATP be moved out of the cell
by coupling it to the downhill movement of Na. Ca moves against the gradient and Na moves in the direction of the gradient
142
define symport and antiport transport
symport - molecules go in the same direction
| antiport - molecules go in the opposite direction
143
describe the ER
it forms an interconnected network of flattened, membrane enclosed sacs or tube like structures known as cisternae. it is continuous with the outer nuclear membrane
144
in plants where else can Ca be stored other than the ER
the vacuole
145
Ca is pumped in from the cytosol to the ER by ………….., an ATPase
SERCA
146
what happens when Ca enters the ER
it binds storage proteins (calsequestrin) so that high Ca concentrations can be achieved. each protein binds ~40 Ca allowing for high concentration in the ER which enables steep [Ca] gradients
147
what is the function of Ca channels
they allow for rapid concentration change (many ions can flow through in a short period) due to steep gradients
148
what is faster, Ca channels or antiporters
channels - Ca is pumped out more slowly than it is allowed in
149
some GPCRs trigger elevations in Ca, what is this mediated by
it is mediated by causing transient opening of a Ca channel in the ER
150
Ca signalling can be activated by ……….. ……….... …….
protein kinase C
151
describe the key features of GPCR signalling
a key feature is activation by phospholipase C by a GTP-binding protein (G protein).
the signal is received by GPCR and the activated G protein subunit activates phospholipase C triggering amplification. phospholipase c breaks down phosphatidylinositol 4,5 bisphosphate into DAG and inositol 1,4,5 trisphosphate (IP3) which activates PKC. due to the conformational change by allosteric modification IP3 opens the ion channel in the ER. the released Ca into the cytosol binds to and activates PKC. Ca and DAG activate PKC
152
what is the difference between tri and tris phosphate
tri - 3 phosphates arranged in series
| tris - 3 phosphates arranged different
153
describe the basic structure of the PKC family
regulatory domain - N terminal, pseudo substrate domain, C1A and C1B, C2, V3 hinge
catalytic domain - kinase domain, C terminal
154
what happens when Ca and DAG bind to the PKC
they induce conformational changes that activate the kinase domain leading to signal transmission
155
what does Ca do to PKC
it binds to C2 and changes the PKC shape and encourages its binding to the PM by linking the negatively charged lipid-phosphatidylserine
156
what does DAG do to PKC
it binds to C1B causing further conformation change and the kinase then activates and is localised to the PM
it also pulls all the other domains away from kinase so it can performs its catalytic activity
157
are both DAG and Ca required for PKC activation
yes
158
why does the PKC kinase need to become membrane localised
because DAG is membrane localised
159
what is the pseudo substrate peptide
it is a peptide that when sitting in the chain, the whole protein is inactive
160
describe beta cell vesicle exocytosis
insulin vesicles are released in response to increased blood glucose
glucose entry changes the ATP/ADP ratio which closes an ATP sensitive channel leading to membrane depolarisation and opening of voltage gated Ca channels and an influx of Ca from the ER or extracellularly. this leads to fusion of the insulin vesicles with the PM and they release their con\tent
161
describe how Ca channels are involved in neural transmission
Ca voltage gated channels are opened by depolarisation signal which is sent along the neuron causing the synaptic vesicles to fuse with the PM, releasing neurotransmitter. this results in a very quick response
162
describe the molecular changes to the Ca channel on opening
- in the closed state the molecules are twisted together
| - upon opening there is a change from isoleucine --> glutamine which are negative, allowing Ca to pass through
163
Ca channel blocker and ACE inhibitors are very common prescriptions for …………..
hypertension - they slow the heart beat so that the left ventricle fills completely. heart workload is reduced which reduces arterial constriction and lowers bp
164
can drugs target selectively for particular Ca channels
yes
165
how does verapanul act
plugs pore preventing Ca entry
166
how does amlopdipine act
remodels channel from outside so Ca lodges inside and prevents conformation change and channel opening
167
how can we monitor Ca changes
FRET
168
describe FRET
we need 2 different fluorescent proteins - CFP (blue) and venus (yellow). CFP is excited at 405nm causing it to emit fluorescence at 447nm. the 2 proteins have spectral overlap. venus can be activated by light from CFP if within 10nm of each other. we can create a construct with 2 fluorescent proteins connected by a hinge molecule which can change its conformation dependent on a Ca signal. when Ca is present the proteins are brought together and we get FRET transfer --> yellow light emitted. we can calibrate the system to measure Ca concentration in cells
169
explain the time differences between insulin secretion which only takes a few seconds and signalling via PKC to takes hours
PKC signalling results in a change in gene expression which takes longer
170
what is phosphorylation
a type of reversible covalent modification involving the transfer of gamma P from ATP onto a protein
171
what is the difference between motifs and domains
motifs tend to suggest functional characteristics and are found within domains. domains tend to relate more to structural units
172
which kinases are the best studied
ser/thr and tyrosine kinases are best studied. there are also histidine kinases but these are rare
173
how is kinase involved in blood glucose control
PKA phosphorylates glycogen synthase switching it off and glycogen phosphorylase switching it on in response to glucagon. we see a 3 kinase pathway. the first kinase activates the second, second activates the third which phosphorylates glycogen and breaks it down. we see huge amplification
174
when the R2C2 complex dissociates is the catalytic subunit immediately active
no
175
where in PKA does ATP sit
in the active site
176
what is the kinase mechanism that is conserved across all kinases
1. ATP binds the active site, substrate binds at the active sites
2. gamma P is transferred from ATP to ser/thr/tyr
3. substrate released from the kinase and so is ADP
177
describe the kinase conserved sequence motifs and differences
- most have GxGxxGx. they all have the gate keeper lysine and the DGF motif
- some kinases have greater distance between motifs than others and motif position in linear sequence is not always indicative of 3D structure
- DGF and gate keeper always end up in the same position in the active site maintain catalytic function
- the substrate and binding site are particular to the target protein
178
describe how kinases can be activated by removing the activation loop and unmasking the active site
the loop block access to the kinase active site so it is inactive (ATP can access but the substrate cant). when the loop is phosphorylated there is a conformational change which leads to the activation loop to move out of the active site. this is known as the priming event. loop sequences have a lot of phosphoacceptor sites. aspartate residues can be catalytic and often closely associated with the target peptide
179
describe the effect of pseudo substrate domains on the kinase active site
the pseudo substrate domain is a small domain that mimics the substrate. it sits in the binding (catalytic) domain and masks it. it has no phosphoacceptor sites. DAG and C1 domain interaction pulls the pseudo substrate out of the active site, activating the kinase
180
what are the different classes of PKC
there are 3 classes - c, n and a
181
how can we predict other phosphoacceptor sites in other proteins
sequences around the substrate phosphoacceptor sites show similarity allowing prediction of other sites in other proteins using bioinformatics - can search for motifs and predict targets
182
what 4 things can phosphorylation alter
change localisation, interactions, half-life and sensitivity to signal
183
what are the 2 main functions of kinases
signal amplifiers and/or key control steps
184
what is Herceptin
an Ab of the EGF tyrosine kinase
185
insulin receptor has an intrinsic/extrinsic tyrosine kinase
intrinsic
186
what is c-src
the first protooncogene - tyrosine kinase
187
why are tyrosine kinases often involved in disease
dues to their function in cell signalling, growth/cell division, metabolism etc
188
describe insulin receptor signalling
the receptor is an alpha2beta2 polypeptide held together by disulphide bonds. insulin binds to the alpha2 subunit inducing a conformational change which results in transmission of the signal to beta2 subunits which leads to the intrinsic kinase in the cytosolic domain being activated. the kinase phosphorylates receptor residues (autophosphorylation) and these residues recruit signalling molecules e.g. IRS-1 which is then also phosphorylated. this all forms a signalling complex
189
describe ligand binding receptor activation
dimersation is key to receptor activation.
EGF, SCF and nerve growth factor exist as monomers then dimerise upon binding their ligand (sometimes required for signal transduction)
insulin receptor is unusual in that it is already a heterodimer
190
it is difficult to get an X ray crystallography of a membrane protein, what can we do instead
we can use an artificial system and cryo EM to study the insulin receptor for example
191
describe cryo EM
samples are frozen to maintain structures then normal EM is carried out
192
what makes the autophosphorylation in the insulin receptor more likely
the huge conformation change brings the kinase domains of the receptor closer together
193
all receptor tyrosine kinases exhibit ………. dependent activation of tyrosine kinase
ligand dependent
194
when the receptor is phosphorylated, the …………. ……………. changes shape so that the kinase domain can be accessed. when the ……... tyr residues are phosphorylated, the loop is flipped open and the active site is available
activation loop
| 3
195
what is trans autophosphorylation
the dimeric receptor phosphorylates trans - chain A phosphorylates tyrosine in chain B and vice versa
196
p tyr act as docking sites for signalling molecules - explain
they allow proteins like SH2 domain containing proteins to be recruited and dock allowing the assembly of signalling complexes
197
different receptors recruit the same/different SH2 domain containing proteins
different
198
why do all SH2 domains exhibit the same shape
because they all have the same arrangement of alpha helix and beta sheet
199
how do SH2 domains show specificity
they all bind p-tyr but show specificity in the 2 pronged concept. they have a p-tyr pocket and a specificity pocket. the surrounding sequence dictates which SH2 domain containing proteins a receptor can bind
200
describe overall signalling complex assembly
ligand binds receptor --> dimerization --> activation of tyrosine kinase domain by release of activation loop --> autophosphorylation --> SH2 domain containing proteins recruitment
- ligand dependent assembly of signalling molecules localised to the receptor and activated with precise spatial and temporal coordinates. different receptors assemble different protein complexes depending on which effectors are recruited.
201
describe leptin signalling
leptin is a hormone that triggers a series of signalling events. the leptin receptor homodimerizes which results in recruitment of JAK2 tyrosine kinase which binds to the dimer a phosphorylates its tyrosines. p-tyr residues recruit STAT3 and allows dimerization and access to the nucleus to act on gene expression
202
why is it important to control signalling pathways
to ensure they are only carried out at the correct time
203
give 2 examples of SH2 domain containing proteins
Ras-GAP, PLCgamma
204
describe PLCgamma activation by RTKs
PLCgamma is recruited via specific SH2 domain. this brings the PLCgamma into close proximity to the tyrosine kinase domain of the receptor
205
what is Ras and what does it do
a g protein and Ras-GTP activates a signalling pathway
206
what is the Ras-GTP/Ras-GDP balance controlled by
relative activity of Ras-GAP and SOS proteins. recruiting these to receptors via SH2 domains is a key control step
207
ras is often mutated in certain …….. ……….
tumour types
208
describe how ERK is produced
as a result of the EGF receptor signalling pathway through Ras --> Raf --> Mek --> Erk
209
how can the distribution of Erk be studied
using immunofluorescence
210
how many genes code for the GPCR receptor superfamily
791
211
what is the beta-2-adrenoceptor the target for
beta blockers and anti-asthma medicines
212
what are GLP-1 mediated functions regulated by
hormones and neurotransmitters
213
where is GLP-1 found and why
in the cells of the gut so it can respond to food availability. it is also found in the pancreatic islets and regulates insulin release, beta cell proliferation and glucagon secretion
214
where is the GLP-1 receptor found
they are found on the beta cells of the pancreas and on the neurons in the brain
215
the GLP-1 receptor regulates systems in the brain, give 2 examples
satiety and control of gastric emptying
216
targeting of the GLP-1 receptor is the basis of a number of treatments of ……...
T2D
217
GLP-1 is a peptide hormone and is destroyed rapidly by …….…., how can these be applied medicinally
DPP4
| DPP4 inhibitors and GLP-1 analogues are used as a medicinal approach
218
why is it a medical issue that many GPCRs are closely related and expressed in multiple tissues
it is associated with side effects and toxicity of treatments
219
what is tiotropum
a muscarinic (acetylcholine) M3 receptor antagonist used to treat COPD but produces unwanted side effects on salvation and in the intestines
220
what can be regulated using selective GPCR drugs
COPD, glucose homeostasis and insulin restriction, detrusor contractibility, smooth muscle contraction, emesis, insulin homeostasis, ageing and dementia
221
what is meant by GPCRs being referred to as serpentine
they are 7 TM receptors - 2 predominantly hydrophobic TM parts
each section is a single polypeptide
serpentine structure is the tertiary structure
222
why do mice have double the GPCRs compared to humans
many of their extra ones control their acute sense of smell. ours became pseudo genes when we became bipedal
223
for signal transduction what must a GPCR interact with
heterotrimeric guanine nucleotide binding proteins
224
how do agonists and inverse agonists affect GPCRs
the receptor is in an equilibrium of inactive and active states and the agonist changes the equilibrium. the agonist allows more active state and the inverse agonist induces the inactive state of the receptor
225
GPCRs can form quaternary structure to control ………. …….…….
activity levels
226
what does tagging a protein by ubiquination do
it targets it to the proteasome for degradation
227
why are there less G proteins than receptors
a range of receptors send equivalent signals
228
what are the 4 G protein groups and their functions
Gs - stimulate adenylyl cyclase and production of cAMP
Gi/o - inhibit adenylyl cyclase to lower cAMP and control ion channels
Gq/11 - stimulate phospholipase C and regulate Ca levels
G12/13 - regulate the cytoskeleton
229
describe the G protein resting state
the G protein is a heterotrimer - 3 subunits - alpha, beta and gamma
230
describe the active state of a G protein
the alpha subunit has GTP bound
231
the ……….. promotes the off rate of GDP from the subunit. GDP is replaced by ……….. causing a physical dissociation of the …… subunit from the other 2
agonist
GTP
alpha
232
to which subunit of the G protein do GDP/GTP bind
the alpha subunit
233
the G protein activation cycle controls what
cAMP production
234
the alpha subunit of the g proteins has an intrinsic ……..
ATPase
235
what does GTPase do
it hydrolyses the last phosphate of GTP and restores GDP to the binding site. the speed of the enzyme process defines how long the 2nd messenger system is being regulated/activated
236
what happens to the G protein upon GDP binding
it allows re-association of the subunits and the protein is ready for the next hormonal signal
237
describe how g proteins are involved in cholera
GTP is not hydrolysed due to toxin inhibiting GTPase. cells continue to make cAMP even when they aren't meant to. ion channels in intestinal cells have their g proteins continually active causing regulation of ion channels in cells causing water to be continually pumped out - diarrhoea
238
how can one recover form cholera if its toxin is an irreversible inhibitor
the intestinal cells turn over quickly so dead cells are removed and new cells are healthy so long as clean water is consumed --> recovery
239
what activates GTPases
guanin nucleotide exchange proteins - blocked in cholera
240
what do adenylyl cyclases do
when they are active they convert ATP --> 3'5' cyclic AMP
241
where does the cyclase enzyme need to be in order to receive a signal and interact with the receptor
at the PM
242
there are ………….. TM proteins that convert ATP to cAMP creating downstream responses (stimulates kinase cascades based on PKA)
12
243
cAMP is polar/non-polar and charged/uncharged so will/wont diffuse out of the cell in a concentration dependent manner
polar
charged
wont
244
what are needed to destroy cAMP
cAMP phosphodiesterases
245
describe the structure of cyclic nucleotide phosphoesterases
N --> C : targeting, regulatory, catalytic, regulatory domains
246
how does the cyclic nucleotide phosphoesterase know where to be active
we want different cAMP concentrations in different cells - there is a sequence in the enzyme that tell it the location to be active
247
which site do all phosphodiesterases have
the catalytic site
248
what is the 2nd messenger of G proteins
cAMP
249
what are the basic steps in drug discovery and development
identify disease, isolate protein involved in disease, find effective drug agent against disease protein, preclinical testing, scale up, formulation, human clinical trials, approval - in total takes around 15 years
250
what is the definition of a hormone
a chemical messenger released by one cell or tissue that, following transport through and aqueous system, alters physiology or response of a different cell or tissue
251
the gut microbiome makes simple molecules from …….……..
dietary fibre
252
what are FA
carboxylic acid with an aliphatic tail which can be saturated or unsaturated
253
cancer, CVD and diabetes are 3 global big killers, what underpins them all
inflammation
254
how does our diet affect the gut microbiome
balanced nutrition, high fibre diet leads to gut microbiota symbiosis and the person is healthy
high fat, processed carbs diet leads to gut microbiota dysbiosis and this can result in obesity/diabetes/inflammatory disorders
255
how do gut microbiota produce large amounts of short chain FA
they ferment fibre and other poorly digested carbohydrates which produces large quantities SCFA
256
how can we mimic good gut microbiota
approaches in humans - faecal transplants, pre/probiotics, antibiotics - all require long term lifestyle changes
beneficial microbial effects independent of microbiota - mimic actions of signalling molecules released by microbes
257
what is photomorphogenesis
when light regulates plant development
258
how do seedlings develop in the light
all resources go into leaf development and chloroplast development
259
how do seedling develop in the dark
extension of hypocotyl
260
how and what do plants detect about light
they sense light spectral quality, quantity, direction and duration using several photoreceptors. plants can detect far lower light intensity than humans
261
what is the UVB photoreceptor
UVR8
262
what are the UVA/blue photoreceptors
cryptochromes and Phototropins
263
what are the red/far red photoreceptors
phytochromes
264
how do photoreceptors detect light
they detect specific wavelengths of light using chromophores (small organic molecule) of various types bound to an apoprotein
265
describe the UVR8 mutant and WT
the mutant is susceptible to damage by UVB radiation which is naturally present in sunlight. In WT UVR8 stimulates expression of genes that protect the plant against UV damage e.g. sun screen compound synthesis in the dermal layer. UVR8 also regulates developmental processes e.g. stem extension growth through regulation of gene expression. the mutant cannot suppress extension growth. UVR8 stimulates rapid extension of a TF called HY5 which stimulates genes in the UVR8 pathway e.g. expression of sunscreen proteins
266
white light does not contain ………...
UVB
267
describe the role of HY5 and its mutant
its function is not restricted to UVR8
HY5 mutants have a long hypocotyl phenotype in all light qualities indicating that it acts downstream of several photoreceptors i.e. UVR8. cryptochrome, and phytochrome to control expression of many genes
268
describe the role of phytochrome
it controls seed germination, stem extension, leaf development and flowering time
269
the phytochrome apoprotein binds a chromophore called ……………….. that absorbs principally red and far red light
phytochromobilin
270
what are the 2 photo-interconvertible forms of phytochrome
Pr and Pfr
271
which of the 2 forms of phytochrome initiates the biological responses
Pfr
272
phytochrome responses are ……….. ……… inducible
red light
273
if far red light is given immediately after red light what happens to phytochrome
Pfr converts back to Pr before the response is formed
274
what initiates phytochrome signal transduction
chromophore isomerises depending on the light quality. this causes a conformation change (twist) to the protein that initiates signal transduction and conversion between the active and inactive form
275
what phenotype do phytochrome mutants have
they have long hypocotyls in red and/or far red light but normal short hypocotyls in other light qualities
276
why cant the HY1 mutant make functional phytochrome and what is the effect of this on the phenotype
they cannot make functional phytochrome due to a defect in chromophore biosynthesis. this leads to the plants having long hypocotyl, less expanded leaves and impaired chloroplast development
277
describe the steps in phytochrome signalling
- after conversion to Pfr, Pfr moves from the cytoplasm to the nucleus
- Pfr then associates with TFs called PIFs, causing their proteolytic destruction
- PIFs repress transcription of genes involved in responses to light, so destruction of them allows stimulation of transcription of target genes
overall: red light --> Pfr --> Pfr PIF --> PIF proteolysis --> transcription. phytochrome gets rid of the repressors allowing photomorphogenesis to occur
278
explain why PIFs accumulate in shaded plants and what does this result in
vegetation absorbs red and blue light but reflects far red light. shaded plants experience lots of far red light (low red : far red) compared to unshaded plants. shaded plants have lower Pfr:Pr which allows PIFs to accumulate leading to the extension growth response due to auxin biosynthesis
279
explain briefly how vertebrate vision works
light entering through the pupil is focussed on specialised light sensitive neurone in the retina: rods and cones. electric signals go into the optic nerve and into the brain
280
describe rods in terms of light sensitivity and colour
high light sensitivity but no colour discrimination
281
describe cones in terms of light sensitivity and colour
less sensitive to light but confer colour vision - they produce different photoreceptors
282
what is the function of ganglion neurons
they gather electrical information from the rods and cones and relay it to the brain via the optic nerve
283
describe the structure of rods and cones
outer segment - membranous discs with rhodopsin photoreceptors
inner segment - nucleus and mitochondria that provide energy for photo-transduction
synaptic part connects to the optic nerve
284
describe rod/cone ion channels
outer segment has an inward Na/Ca channel gated and opened by cGMP
the inner segment has an Na/K pump
285
how does light perception affect the ion channels of rods/cones
it leads to a decrease in cGMP concentration in the cytoplasm which closes the Na/Ca channel causing a change in membrane potential which is transmitted through the neuron
286
what is the visual photoreceptor
rhodopsin
287
what is the structure of rhodopsin
consists of opsin protein with retinal chromophore. opsin has 7TM alpha helices
288
what happens when retinal absorbs light
it undergoes isomerisation which causes conformational change to opsin
289
why are there 2 photoisomers of rhodopsin
due to the rotation around the carbon atom
290
what are the similarities between rhodopsin and channelrhodopsin
7 TM structure and retinal chromophore
291
what are the differences between rhodopsin and channelrhodopsin
opsin doesn't form an ion channel and retinal has different light induced isomerisation. rhodopsin: 11-cis --> all-trans isomerisation. channelrhodopsin: all trans --> 13 cis isomerisation
292
rhodopsin is associated with g protein called …………..
transducin
293
what are the steps in rhodopsin photoreception
photoreception causes exchange of GDP --> GTP on the transducing alpha subunit and dissociation of the alpha subunit
activated transducing alpha stimulates cGMP phosphodiesterase by removal of an inhibitory subunit.
this results in cGMP --> 5'-GMp lowering [cGMP]
this leads to closure of cGMP gated Na/Ca channels, causing a change in membrane potential from -40 --> -75mV in the light
this stimulates a signal through the optic nerve
294
describe the desensitization that occurs after rhodopsin photoreception
continued illumination of rhodopsin exposes ser/thr amino acids that are phosphorylated by rhodopsin kinase. P-rhodopsin is bound by arrestin 1 which prevents further interaction with transducing and stops signalling.
295
how does colour vision work
each type of cone expresses a different opsin, although they are all closely related in sequence. the retinal is in different molecular environments in the cone opsins, giving different absorption spectra
296
what do mutations in opsins lead to
colour blindness
297
what is melanopsin
the 3rd type of photoreceptive neuron in the retina, not involved in vision. intrinsically photosensitive retinal ganglion cells contain melanopsin as the photoreceptor. melanopsin binds retinal and is maximally sensitive to blue light. it activates a G protein by a different mechanism to transducin and resembles invertebrate opsins in structure and signalling. it functions in entrainment of circadian rhythms and behavioural responses to light
298
what is low melanopsin associated with
seasonal affection disorder (SAD) - winter depression
299
what is optogenetics
using light to understand complex signalling networks
300
describe neurons
they are the basic unit of the nervous system. they send and receive signals, the cell body contains the nucleus, dendrites are the receiving part of the cell, axons send information on. cells are electrically excitable and vary in length
301
describe the resting state of a neuron
the neural membrane is polarised - positive on the outside and negative on the inside
302
describe the generation of an action potential (overall)
when neurons receive signals, protein channels open in the membrane resulting in localized influx of Na which leads to depolarisation (reduction of charge difference across the membrane) which activates neighbouring Na channels creating a wave of depolarisation
303
what are the 5 steps in the action potential
1. Na channels open
2. more Na channels open
3. Na channels close
4. K channels open
5. K channels close
304
the wave of depolarisation starts at the ……. and moves towards the ………. ………… through the …...…. to its terminal
dendrite
cell body
axon
305
what happens at the synapse during neural transmission
depolarisation at the axon terminus/synapse triggers release of neurotransmitters which diffuse across the synapse and stimulate electrical excitation of the next cell.
306
what do excitatory neurotransmitters do
they initiate depolarisation through opening of Na channels
307
what doo inhibitory neurotransmitters do
they lead to opening of Cl channels and increase polarization, decreasing the chance of depolarisation
308
why are negative ions inhibitory in neural transmission
because they cause the wrong charge change
309
what can electrical stimulation be used to treat
Parkinson's, depression etc but there are unwanted side effects due to the lack of specificity
310
what is the disadvantage of chemical stimulation treatment compared to electrical stimulation
chemical stimulation takes longer
| it also shows side effects
311
why is optogenetics useful
light is easy to manipulate, can be delivered spatially and produces fast effects. we can use light sensitive proteins or photoreceptors which can be used to activate/inactivate neurons
312
what is Chlamydomonas reinhardtii
it is a unicellular green algae that s reactive to sunlight. they grow in aquatic environments and move towards the light to maximise capture for photosynthesis. they have a light activated channel that is used to control movement
313
is channelrhodopsin a GPCR
no - it is a blue light activated single component system that transports positive ions into the cell (primarily Na)
314
why does channelrhodopsin respond to light
due to rhodopsin and channelrohodopsin cofactors in each of the 7 TM proteins.
315
channelrhodopsin controls ...…...…., it makes animal cells respond electrically to light (it is a light activated …....…. channel)
phototaxis
| cation
316
neurons can be made electrically responsive to blue light by using ………...….... in optogenetics. the neurons recover in darkness
channelrhodopsin
317
what is halorhodopsin
a light activated Cl pump found in algae and archaebacteria.
318
what colour of light drive Cl influx by halorhodopsin and why
yellow - retinal absorbs yellow because of the changed protein environment
319
opening of Cl channels increases polarization/depolarization and can activate/inhibit neural activity
polarization
| inhibit
320
how can we make a synthetic light activated GTPase to spatially control cell shape/proliferation
fuse DNA encoding photoreceptor domain, LOV flavin chromophore, DNA encoding animal GTPase.
LOV binds a flavin derived cofactor which absorbs UVA blue light. can take molecule out and fuse to protein of choice - it will hydrolyse GTP --> GDP creating an artificial photoreceptor system. in light GTPase is active and in dark it is disrupted.
light activated GTPase stimulates change in cell shape
