D. Basic Molecular Biology - Signalling Flashcards
What is GPCR signalling? (2)
- More than 1000 different GPCRs
- Regulate smooth and cardiac muscle contraction, kidney function, smell,
neurotransmission
- ______GPCR signalling plays a role in obesity, cardiovascular disease, inflammation, diabetes, depression and alzheimer disease
- ____% of marketed drugs target these receptors of pathways they activate
Faulty
50%
GPCR signalling:
- Family of integral membrane proteins with 7 _____-_________ proteins while linked to a _______ guanine nucleotide-binding protein (G protein)
- 7 transmembrane receptor/serpentine receptor
- ______ binding to GPCR receptors activated G proteins
spanning proteins
ligand
What is Gs alpha subunit
(Gs GPCR SIGNALLING) ? (3)
- activated by hormone binding to receptor
- Gs subunit diffuses along membrane activating
adenylate cyclase - Converts ATP to cAMP which activates PKA
which phosphorylates target proteins
● Gs =
● Gs = stimulatory, activating (A -> proteins with a in them )
What is the Olfactory signalling pathway? (4)
- using GPCR
- Odorants binds to receptor
- Adenylate cyclase activated and cAMP produced
- Increase in cAMP opens channels that permit Na and Ca2+ entry into the cell and depolarise it
What is the Gq GPCR SIGNALLING pathway? (3)
- phospholipase C cleaves PIP2 into DAG and IP3
- Second messengers activate kinases
(ca/calmodulin dep kinase and PKC) - Phosphorylation of target proteins
What is the Gq GPCR SIGNALLING pathway? (3)
- phospholipase C cleaves PIP2 into DAG and IP3
- Second messengers activate kinases
(ca/calmodulin dep kinase and PKC) - Phosphorylation of target proteins
● Gq=
Contains a lot of C
What is the Taste signalling pathway? (3)
- using GPCR
- Sweet, bitter or umami ligand bind to taste
GPCRs and activate phospholipase C which
produces IP3 - IP3 elevates cytoplasmic Ca2+ and depolarises the membrane via cation channel
What is Receptor tyrosine kinase (RTK) signalling? (4)
- more than 60 known receptor tyrosine kinases which are involved in cellular signalling pathways
- Regulate key cellular processes such as proliferation, differentiation, development, angiogenesis
and survival - Unregulated activation of this pathway can lead to cancer as well as benign proliferative conditions
- plays a role in inflammatory diseases and diseases
Receptor tyrosine kinase (RTK) signalling
- Targeted molecular therapies against members of RTK receptor family are amongst first in use: (3)
- Trastuzumab: Herceptin (anti-HER2) > used in breast cancer
- Gefitinib: anti-EGFR > used in lung cancers
- Many others are presently used and being developed
What are RTK: Ligands/first messengers?
- regulates cellular responses to growth factors (GF receptor signalling pathway)
What are the types of
RTK: Ligands/first messengers? (5)
- Epidermal Growth Factor (EGF) > produced by many different cells and contains hydrophobic
domain allowing it to be anchored to plasma membrane. When required in wound healing, it is
cleaved from membrane and functions to signal cell proliferation - Platelet derived growth factor (PDGF) > crucial roles during development. Produced by platelets,
smooth muscle cells etc. Some secreted in inactivated form and cleaved by proteases in ECM and
activated - Fibroblast growth factor (FGF) > important in control of development
- Vascular endothelial growth factor (VEGF) > major role in angiogenesis
- Angiopoietins > controls angiogenesis. Important in cell adhesion of haematopoietic stem cells to
stem cell niche
What are the types of
RTK: Ligands/first messengers? (5)
- Epidermal Growth Factor (EGF) > produced by many different cells and contains hydrophobic
domain allowing it to be anchored to plasma membrane. When required in wound healing, it is
cleaved from membrane and functions to signal cell proliferation - Platelet derived growth factor (PDGF) > crucial roles during development. Produced by platelets,
smooth muscle cells etc. Some secreted in inactivated form and cleaved by proteases in ECM and
activated - Fibroblast growth factor (FGF) > important in control of development
- Vascular endothelial growth factor (VEGF) > major role in angiogenesis
- Angiopoietins > controls angiogenesis. Important in cell adhesion of haematopoietic stem cells to
stem cell niche
- Heparan sulphate proteoglycans play an important role in ____ _______ by binding GFs such as FGF and PDGF (restricting their action to areas close to site of release)
- Play important roles in ____ ______ and if overactive can result in cancer
cell signalling
wound healing
RTK signalling
- Monomer proteins: (4)
- External domain > binding to ligand
- Transmembrane domain
- Catalytic domain
- Tyrosine amino acids in the tail
- Upon binding of ligand to RTK receptor, ______ and conformational changes occur which activates the intrinsic tyrosine kinase activity of receptor and phosphorylates______ residues within cytoplasmic portion
- Either mitotic activated protein kinase (______) or Phosphoinositide-3-kinase (_____) pathways or both may be activated
- ____ recognises phosphorylated tyrosine and aa surrounding
- MAPK
- Adaptor SH2 binds - PI3K
- PI3K SH2 domain binds
- Both pathways can be initiated by ligand binding to _____ receptor
dimerisation
tyrosine
(MAPK)
(PI3K)
RTK
Receptor tyrosine kinase signalling: MAPK
What is the full process? (9)
- ligand binds to inactive monomers causing dimerisation
- RAS is anchored to the membrane, and attached is GDP (inactive)
- Phosphorylated tyrosine is recognised by SH2 domain (adaptor protein)
- GEF binds (has domain that interacts with adaptor protein)
- GEF exchanges GDP on RAS for GTP (activated)
- RAS initiates a MAPK cascade (each kinase
initiated by phosphorylation) - MAPK is phosphorylated and activated
- It can phosphorylate proteins (to change function) or TFs (to activate)
- Results in proliferation
Receptor tyrosine kinase signalling: MAPK
What is the full process? (9)
- ligand binds to inactive monomers causing dimerisation
- RAS is anchored to the membrane, and attached is GDP (inactive)
- Phosphorylated tyrosine is recognised by SH2 domain (adaptor protein)
- GEF binds (has domain that interacts with adaptor protein)
- GEF exchanges GDP on RAS for GTP (activated)
- RAS initiates a MAPK cascade (each kinase
initiated by phosphorylation) - MAPK is phosphorylated and activated
- It can phosphorylate proteins (to change function) or TFs (to activate)
- Results in proliferation
What is RAS? (4)
- Small monomeric G protein (RAS)
- GEF activates RAS (by exchanging guanine nucleotides)
- Intrinsic GTPase activity by removing terminal/gamma phosphate
- Results in inactive RAS with GDP
What is PI3K signalling? (10)
- Initiated by growth factors binding to RTKs
- Binding of ligand to receptor causes dimerisation, shape
change and activation of catalytic domain - Phosphorylated tyrosine tail
- PI3K, SH2 domain binds to tail
- PI3K is linked to receptor (close to membrane) and
phosphorylates PIP2 converting it to PIP3 - PH domain of AKT interacts with PIP3 on membrane
- AKT gets phosphorylated and activated
- AKT moves back into the cytoplasm and phosphorylates proteins
- AKT does not affect transcription, changes protein function
- Phosphorylated proteins regulate cell survival (inhibits apoptosis) and increases cell cycling
What is PI3K signalling? (10)
- Initiated by growth factors binding to RTKs
- Binding of ligand to receptor causes dimerisation, shape
change and activation of catalytic domain - Phosphorylated tyrosine tail
- PI3K, SH2 domain binds to tail
- PI3K is linked to receptor (close to membrane) and
phosphorylates PIP2 converting it to PIP3 - PH domain of AKT interacts with PIP3 on membrane
- AKT gets phosphorylated and activated
- AKT moves back into the cytoplasm and phosphorylates proteins
- AKT does not affect transcription, changes protein function
- Phosphorylated proteins regulate cell survival (inhibits apoptosis) and increases cell cycling
What is the WNT pathway? (3)
- important role in embryogenesis of colorectal epithelia
- Important for bone formation and remodelling in human joint disease eg rheumatoid arthritis
(inhibited pathway) and ankylosing spondylitis (overactive pathway) - Modulation of pathway by gene therapy to treat arthritis is underway
What is the WNT pathway (with no ligand)? (8)
- processes still occur without ligand
- 2 receptors in membrane: frizzled and LRP
- In cytoplasm, protein complex formed: degradation complex: Axin, B catenin, GSK and APC
- APC and axin are scaffold protein
- GSK phosphorylates B catenin, changing the B
catenin shape - Now recognised by E3 ligase
- E3 ligase attaches chain of ubiquitin, allowing B catenin to be recognised by the proteasome and degraded
- No transcription of genes occurs and no gene expression
What is the WNT pathway: ligand present? (8)
- WNT binds to receptors, bringing them together and
changing their shape - Axin binds to tail of LRP
- GSK is inhibited by series of reactions from frizzled
receptor - Degradation complex not formed
- B catenin present in cytoplasm
- If B catenin is non phosphorylated, it will not be
ubiquitinated and not degraded by proteasome - B catenin levels increases and moves into nucleus,
binds to T cell factor and initiates transcription of genes - Leads to proliferation and production of G1 cyclin protein
What is the WNT pathway: ligand present? (8)
- WNT binds to receptors, bringing them together and
changing their shape - Axin binds to tail of LRP
- GSK is inhibited by series of reactions from frizzled
receptor - Degradation complex not formed
- B catenin present in cytoplasm
- If B catenin is non phosphorylated, it will not be
ubiquitinated and not degraded by proteasome - B catenin levels increases and moves into nucleus,
binds to T cell factor and initiates transcription of genes - Leads to proliferation and production of G1 cyclin protein
What is the cell cycle?
- sequence of events resulting in growth and division of cells into identical daughter cells
What are the Cell cycle requirements? (4)
- double cytoplasm and organelles
- Safely and faithfully duplicate DNA
- Separate cellular content
- Check the integrity of the process and correct any errors
What are the phases of the cell cycle? (7)
- GO > quiescent, resting, non proliferative phase > not
going through cell cycle - G1 > Gap1 phase, 9 hrs long, regular metabolic activities
and preparing for the next phase of cycle, signal committing the cell to replicate is received here. If division signal is not received, cell enters a quiescent G0 phase - S > synthesis phase, 10 hrs, DNA is replicated to produce 2 daughter chromatids
- G2 > Gap2 phase, 4 hrs, proteins synthesised and grows in size
- M > mitosis, 1 hr, chromosomes divided and cytoplasm splits (2 coupled process) (prophase, prometaphase, metaphase, anaphase and telophase)
- Interphase: G1 + S + G2
- Centrioles replicate between G1 and G2
What are the phases of mitosis?
- prophase > chromatin condenses to form individual chromosomes each consisting of 2 identical
double stranded helical DNA molecules (sister chromatids), nucleolus and nuclear membrane start to disappear - Prometaphase > chromosomes condense further (each forming a kinetochore - to which spindle fibres attach)
- Metaphase > chromosomes align along metaphase plate and 2 kinetochores face in opposite directions and joined by microtubules to opposite poles
- Anaphase > chromosomes separate into 2 sister chromatids and move towards opposite poles by mitotic spindle
- Telophase > sister chromatids reach opposite poles and nuclear membrane starts to form, mitotic spindle breaks down and chromosomes decondense
Duration of cell cycle
- Typically rapidly proliferating cell would = ___ hrs
- Length of time occupied by S, G2 and M is relatively constant
- Length of cell cycle determined by _____ of the G1 phase (non-existent in rapidly dividing embryonic cells, infinite in some differentiated cell)
- Neurons and skeletal muscle fibres cease cell division all together and remain in arrested ____ >
injury to neurons are difficult to repair
- Fibroblasts, liver, kidney and lung cells _____ occasionally (usually in G0 but can be activated by
external agents)
- Cells of skin and lining intestinal tract are exposed to trauma so ______ replaced
24
duration
G0 >
divide
continuously
What is Cell differentiation? (5)
- embryonic cells become different from one another acquiring distinct identities and functions
- Differentiation is usually accompanied by cell cycle exit in G1
- Cell cycle is inhibited by specific proteins that prevent progression from G1 to S
- Differentiated state some genes are active and other repressed
- During process of differentiation, each primary cell type expresses specific proteins particularly
suited to control their particular function
What is Cell differentiation? (5)
- embryonic cells become different from one another acquiring distinct identities and functions
- Differentiation is usually accompanied by cell cycle exit in G1
- Cell cycle is inhibited by specific proteins that prevent progression from G1 to S
- Differentiated state some genes are active and other repressed
- During process of differentiation, each primary cell type expresses specific proteins particularly
suited to control their particular function
Regulation of cell cycle: restriction point
What is this point? (3)
- cell proliferation regulated late in G1 at restriction point
- As a result of actions of growth factors, cell passes restriction point and enters S phase, one
passing the point they are committed to the cell cycle - If the appropriate growth factors are not available, the cycle stops at restriction point and cell enters
G0
Regulation of the cell cycle: cyclins and cyclin dependent kinases
How is regulated?
- molecular events that control the cell cycle are ordered and directional, cycle can not be reversed
- Two key classes of regulatory protein molecules that determine process of a cell through the cycle -
cyclins and cyclin dependent kinases - Cyclins are produced cyclically
- Cyclins and CDKs undergo assembly into complexes, activation by phosphorylation and then
disassembly and cyclins are degraded -> prevents cycle from going in reverse - Controls basic cycle allowing movement from G1 to S, S to G2 and G2 to M
- Cyclins degraded in proteasome
Regulation of the cell cycle: cyclins and cyclin dependent kinases
How is regulated?
- molecular events that control the cell cycle are ordered and directional, cycle can not be reversed
- Two key classes of regulatory protein molecules that determine process of a cell through the cycle -
cyclins and cyclin dependent kinases - Cyclins are produced cyclically
- Cyclins and CDKs undergo assembly into complexes, activation by phosphorylation and then
disassembly and cyclins are degraded -> prevents cycle from going in reverse - Controls basic cycle allowing movement from G1 to S, S to G2 and G2 to M
- Cyclins degraded in proteasome
- G1 and S phase:
- G2 and M phase:
- G1 and S phase: CDK2
- G2 and M phase: CDK1
Regulation of the cell cycle: cyclins and cyclin dependent kinases
G1 cyclin and CDK2 = (3)
- G1 cyclins increase during G1
- Interact with CDK2 and activate it
- Once CDK2 has phosphorylated what’s needed, G1 cyclin decreases
Regulation of the cell cycle: cyclins and cyclin dependent kinases
S cyclins and CDK2/CDK1= (3)
- s cyclin increases at the end of G1 and degraded during G2
- Binds to CDK2 and activates it
- Blinds to CDK1 in G2 and activates it
Regulation of the cell cycle: cyclins and cyclin dependent kinases
M cyclins and CDK1 = (2)
- levels increase I’m G2 and rapidly decreases and
metaphase anaphase junction - Binds to CDK1 and activates it
What are the Cell cycle checkpoints? (5)
- positions where conditions are checked to allow
continuation of cycle (DNA undamaged) - Check the integrity to all aspects and stop cycle
if cell is not perfect - G1/S checkpoint
- G2/M checkpoint
- Metaphase-Anaphase checkpoint also known as
spindle assembly checkpoint
G1/S checkpoint >
G1/S checkpoint > cell cycle arrested in G1 if DNA damage is detected, to allow time for DNA repair. Controlled by p53 (allows DNA repair before entry into S phase)
G2/M checkpoint >
G2/M checkpoint > at the end of G2 before mitosis, checks quality of DNA replication. If DNA is damaged, p53 regulates repair
G2/M checkpoint >
G2/M checkpoint > at the end of G2 before mitosis, checks quality of DNA replication. If DNA is damaged, p53 regulates repair
Metaphase/anaphase checkpoint >
Metaphase/anaphase checkpoint > end of metaphase, checks quality of mitosis machinery (alignment of chromosomes on metaphase plate, chromatid centromere connections to spindles)
What are Apoptosis and necrosis? (3)
- different mechanisms by which a cell can die
- Necrosis (accidental cell death) caused by physical/chemical injury
- Apoptosis (programmed cell death) which is dismantling of the cell in response to extracellular
signals, cellular stress or chemotherapy
What is the process of Necrosis?
- cell membrane integrity is lost (pumps not working correctly)
- Cell and its organelles swell and eventually burst
- Lysosomal enzymes and cytoplasmic material leak into extracellular space
- An inflammatory response is initiated which is detrimental to adjacent cells
- Usually as a result of acute hypoxic or ischemic injury - MI or stroke, supraphysiologic conditions or toxins
What is the process of Necrosis?
- cell membrane integrity is lost (pumps not working correctly)
- Cell and its organelles swell and eventually burst
- Lysosomal enzymes and cytoplasmic material leak into extracellular space
- An inflammatory response is initiated which is detrimental to adjacent cells
- Usually as a result of acute hypoxic or ischemic injury - MI or stroke, supraphysiologic conditions or toxins
Renal ______ necrosis
cortical
What is Apoptosis? (3)
- regulated breakdown of cellular proteins by enzymes
(caspases). A cysteine in active site aids in recognising and cleaving target protein only after an aspartic acid residue - Results is formation of apoptotic bodies (cell fragments surrounded by intact membrane which are engulfed by
macrophages/neighbouring epithelial cells - Unwanted, aged or sublethally damaged cells are removed via a process that does not activate the immune response
DNA laddering in apoptosis:
- endonuclease cuts nucleosomes in ______ region
- Fragments of _____ bp in size (per nucleosome)
- DNA run on agarose gel and separates by number of bp > _____ ______
- In necrosis, many different fragments of different sizes
linker
180
ladder effect
Apoptosis vs Necrosis
Why is Apoptosis important? (2)
- need for proper development > resorption of tadpole tail and formation of fingers and toes in foetus requires apoptosis
- Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism > cells infected with viruses, cells of the immune system (excess cells produced in cell mediated immune response are removed), cells with DNA damage, cancer cells
Apoptosis occurs along 2 pathways:
- extrinsic (death receptor mediated) and
- Intrinsic ( mitochondria mediated)
What is the Extrinsic pathway of Apoptosis? (9)
- receptors in the membrane (death receptors - monomers in membrane)
- Trimerise when death ligand binds (either found as soluble in ECF or on membrane of cytotoxic CD8 T cell
- Death ligand causes trimerisation of death receptor
- Adapter proteins bind (3 could bind)
- Adapter protein binds to initiator pro caspase 8
- DiSC: death inducing signalling complex
- Initiator procaspase 8 auto cleave each other to form initiator caspase 8 (active)
- Cleaves effector procaspases 3,6, 7 to produce effector caspases 3,6,7 (active)
- Cellular proteolysis leading to apoptosis
What is the Extrinsic pathway of Apoptosis? (9)
- receptors in the membrane (death receptors - monomers in membrane)
- Trimerise when death ligand binds (either found as soluble in ECF or on membrane of cytotoxic CD8 T cell
- Death ligand causes trimerisation of death receptor
- Adapter proteins bind (3 could bind)
- Adapter protein binds to initiator pro caspase 8
- DiSC: death inducing signalling complex
- Initiator procaspase 8 auto cleave each other to form initiator caspase 8 (active)
- Cleaves effector procaspases 3,6, 7 to produce effector caspases 3,6,7 (active)
- Cellular proteolysis leading to apoptosis
Intrinsic pathway
- BCL2 family members: (2)
- prosurvival (BCl2: BH1, BH2, BH3, BH4)
- Pro-apoptosis (BAx family: BH1, BH2, BH3) (BH3 only)
BCL2 proteins regulate ____ formation in mitochondria
- anti apoptotic eg BCL2, BCLxl
- Pro apoptotic
- Multi Domain eg. BAX, BAK
- BH3 domain only eg. BAD, BIM
Dimer of BAX or BAK in membrane to form pore which releases cytochrome c -> initiation of _____ pathway
BCL2 binds to BAX, prevents pore formation and cytochrome C formation
- BAD binds to BCL2, allowing BAX to form dimers and create pores and release cytochrome C
pore
intrinsic
What Intrinsic pathway of Apoptosis? (6)
- initiated by chemotherapy or irradiation
- Increased BAX, allows dimer formation, pore
creation and cytochrome c release - Initiator procaspase 9, cytochrome c and
APAF1 form apoptosome - Initiator procaspase 9 cleaves to form active
caspase 9 which cleaves effector procaspase to
produce active effector caspases - Leads to cellular proteolysis and apoptosis
- CD8 T cells secretes granzyme which causes
apoptosis by activating cytochrome C and activating effector procaspases to produce effector caspases
Diseases associated with apoptotic abnormalities Defects of apoptosis: (2)
- autoimmune disease such as lupus and rheumatoid arthritis
- Autoimmune antibody producing B cells not signalled to apoptosis during B cell development - Cancer development as cells continue to survive instead of being removed ones su lethal mutations have occurred eg lymphoma
- Drugs in clinical trials to induce apoptosis
Diseases associated with apoptotic abnormalities
Uncontrolled or overactive apoptosis: (3)
- Alzheimer’s and Parkinson’s
- Excessive loss of uninflected CD4 T cells in HIV and AIDS
- Sepsis - huge apoptosis of immune regulation cells and GIT cells
