Theme 3 : Molecular cell biology Flashcards
What are Transporters? Give an example
Na+ pumps actively pumping Na+ out of cells and K+ in
What are Anchors? Give an example
Example: Integrins
They link intercellular actin filaments to extracellular matrix proteins
Give an exampleof a receptor
Platelet-derived growth factor (PDGF) receptor, binds to PDGF and generates intracellular signals that cause the cell to grow and divide
Give an example of an enzyme incorporated in a membrane
Adenylyl cyclase catalyses the production of cyclic AMP in response to extracellular signals
What does Inhalation of hydrogen cyanide or ingestion of potassium cyanide do?
inhibits the mitochondrial electron transport chain at the cytochrome oxidase step.
Is one of the most potent and rapidly acting poisons known.
What are Mitochondrial myopathies and what is it caused by?
A genetic disorder caused by = Point mutations in genes encoding the mitochondrial tRNA lysine.
This causes myoclonic epilepsy and ragged fibers
Skeletal muscles contain abnormally shaped mitochondria and have decreased cytochrome oxidase activity.
What are Peroxisomes? And what do they do?
Small cytoplasmic vesicles and has a contained environment for reactive H2O2 generation
The main function of the oxidation reactions is the breakdown of fatty acids
Detoxify toxic substances such as ethanol (via catalase)
Cytosol
Contains many metabolic pathways
Intermediate filaments
Part of the cytoskeleton
Lysosomes
Intracellular degradation
What do Endosomes do?
sorting of endocytosed material (the material to be internalised is surrounded by an area of plasma membrane, which then buds off inside the cell to form a vesicle containing the ingested material.)
Functions of the cytoskeleton (5 things)
Pulls the chromosomes apart during mitosis
Drives and guides the intracellular traffic of organelles, proteins, and RNA
Supports the plasma membrane
Enables some cells to move
Controls cell shape
What are the 3 major components of the cytoskeleton?
Intermediate filaments, microtubules, actin filaments
Actin filaments
Polymers of actin monomers
Necessary for movement
Can form contractile bundles and microvilli
May associate with myosin to form powerful contractile structures
Carry cargo-bearing motor proteins (e.g. myosin)
Intermediate filaments
10 nm diameter filaments
Made of a family of fibrous proteins
Twisted into ropes and provide tensile strength
Needed to maintain cell shape
4 types
What fibrous proteins are intermediate filaments made out of?
keratin filaments in epithelial cells
vimentin in many other cells
neurofilament proteins in neurones
lamins within the nucleus
Microtubules
20 nm diameter
Polymers of tubulin dimers
Organised from structures such as the centrosome
Form the spindle in mitosis
Important in cell shape and movement
Carry cargo-bearing motor proteins
describe dynein
0.2- 60 micrometers/sec on the left direction of microtubules - minus end
kinesin
(0.02-2microm/sec) to right direction - plus end
What happens when a stem cell divides
Stem cell detaches its receptor from the ligand of a niche (stromal cell) and commits terminal differentiation or die
Where are adult stem cells made in the gut? Explain how?
Crypt on the Lumen of the gut. Moves up the crypt. Slow dividing stem cells (more than 24 hours). Then rapid
Where are adult stem cells made in the skin? Explain how?
Between the lining of the dermis and epidermis
Advantages of IPS
Cells taken from patient should not elicit immune response
Fewer ethical issues
Theoretically, any cell type could be replaced
Disadvantages of IPS
More basic research needs to be done on developmental pathways
Transplanted stem cells could develop into cancer cells
Describe apoptosis (4 things)
Degrade intracellular structures and organelles
Collapse the cytoskeleton
Fragment the cell into mini-cells, which are engulfed by phagocytes for degradation.
neatly without damaging its neighbours
Necrosis
The cell membrane’s integrity is destroyed.
The cell’s soluble contents are released into the tissue fluids.
Cell components are degraded by the actions of extracellular enzymes and phagocytic cells engulf the fragmentary remains.
Outline Constitutive secretion
modification, packaging and sorting of proteins
Golgi apparatus -> newly synthesised soluble proteins, plasma membrane protein, plasma membrane lipids W/ transport vesicle -> unregulated membrane fusion
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Biomarker
a naturally occurring molecule, gene, or characteristic by which a particular pathological or physiological process, or disease can be identified.
Translational research
It translates findings in fundamental research into medical practice and meaningful health outcomes.
Stratified medicine
identification of subgroups of patients with distinct mechanisms of disease, or particular responses to treatments that allow us to identify and develop treatments that are effective for particular groups of patients.
Precision medicine
medical decisions, practices, interventions and/or products being tailored to the individual patient
Pharmacogenomics
The study of how genes affect the response to a particular drug
Efficacy
the ability, especially of a medicine or a method of achieving something, to produce the intended result
Tumour necrosis factor (TNF)
a cell signalling protein (cytokine) involved in systemic inflammation.
Interleukin-1 (IL-1)
a pro-inflammatory cytokine that can induce inflammation and fever
C-Reactive Protein (CRP)
Used to measure inflammation
Anticyclic citrullinated peptide antibody
Used to help diagnose rheumatoid arthritis
CYP2D6 enzyme
metabolise a wide range of drugs including tricyclic antidepressants and the breast cancer drug tamoxifen. It is highly polymorphic with more than 70 allelic variants
examples of where stratified medicine is being used clinically
Breast Cancer Patients
Cetuximab
An inhibitor of the epidermal growth factor receptor
What makes up a human nucleus?
Nuclear envelope
Nuclear pore
Nucleolus
Heterochromatin
Euchromatin
What is Heterochromatin?
Tightly packed, dense mass of protein and DNA
What is Euchromatin?
DNA and histones
What does the nucleolus do?
transcribes ribosomal RNA
What are the 3 functions DNA must fulfil?
- Encode all info required to make an organism
(DNA to RNA to protein) - It must replicate itself accurately
- It must allow beneficial mutations to be selected
Genetics
the study of heredity, the process by which characteristics are passed from parents to offspring
Gene
is a unit of biological information that encodes a specific protein or regulatory molecule
Why do we look different if 99.9% of our genes are identical?
Each have ~3 million single nucleotide polymorphisms (SNP)
How many SNP’s does each person have that is associated with an inherited disease
~50-100 SNPs associated with an inherited disease
What were the 2 impacts of genomics in medicine?
Functional genomics
Personalised medicine
Explain the 2 impacts of genomics in medicine
F: Mechanisms of disease
Targeted therapeutics and gene therapy (CRISPR)
Human evolution
P: Predictive (Huntington’s disease)
Pharmacogenics
Ethics
What is the structure of DNA?
(3 Basic components)
Double helix of nucleic acid
Complementary strands of nucleotides
Adenine=Thymine
Cytosine=Guanine
Sequence of bases codes for different proteins
What are the distance between backbones in DNA called?
Minor and Major groove
Why does DNA have different strand directions?
Polar molecule
Where are covalent links found and how strong are they?
Between the phosphate group and pentose sugar on the sugar-phosphate backbone
Strong
Where are hydrogen bonds found and how strong are they?
Between nucleotide bases
Weak
What is the covalent bond called? What do they do?
Phosphodiester bonds link nucleotides
What is the outside of DNA composed of?
DNA sequence highly condensed around histone proteins – multiple levels of folding
Describe the first level of folding
DNA to Nucleosome
DNA is wrapped many times around a barrel of 8 histones (octamer)
(2x) H2A, H2B, H3 and H4 molecules
Histones +ve, DNA –ve charged
Describe the second level of folding
Additional protein (H1) allows it to fold back on itself and packages for 30nm fibre strip of chromatin where dna is most of the time transcription to occur
Why is DNA condensation highly regulated?
Allow access to enzymes for DNA replication or gene expression
What are the 2 methods of DNA condensation?
- Chromatin-remodelling complexes
- Histone-modifying enzymes
(add or remove acetyl, phosphate or methyl groups
How can supercoiling affect gene expression?
May cause gene expression to be switched off
Name the consequences of supercoiling if it goes wrong
β-globin: severe anaemia
Tumour suppressor genes: cancer
Epigenetics
heritable changes in gene function that cannot be explained by changes in DNA sequence (e.g. methylation) – impact of environment
What does a typical chromosome consist of? (one of the chromatids)
Heterochromatin
Euchromatin
Telomere
Centromere
What are the components in DNA packaged into chromosomes?
22 pairs homologous autosomal chromosomes
+ 2 sex chromosomes (XX/XY) = 46
When did the Human genome project start?
1990
Human Karyotype use?
Used to diagnose gross genetic changes
What does Cytogenetics do?
Stained with Giemsa to analyse G-banding pattern
What are the phases that are part of the cell cycle?
M phase, G1 Phase, S Phase, G2 Phase
What are the 6 stages of M phase?
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
Cytokinesis
How long is DNA fibre?
30nm
Which phase does DNA replication occur?
S phase
What is the tip of the short arm of a chromosome called?
Telomere
What is the name of the component that is adjacent to the spindle microtubules and centromere?
Kinetochore
Prophase
Chromosomes condense
Mitotic spindles form
Prometaphase
Nuclear membrane disintegrates
Spindles attach to kinetochores
Metaphase
Chromosomes align at equator
Kinetochores of all chromosomes aligned in a plane midway between 2 spindle poles
Anaphase
Sister chromatids separate
Pulled towards spindle poles
Kinetochore microtubule shortens
Telophase
Contractile ring starts to form
Chromosomes arrive at spindle poles
Nuclear envelopes reform
Cytokinesis
Contractile ring creates cleavage furrow
Cytoplasm divides resulting in two genetically near-identical cells
Re-formation of interphase array of microtubules nucleated by the centrosome
What controls cell cycle checkpoints?
Controlled by cyclins and protein kinases (Cdks): phosphorylation of cdk/cyclin complexes
What do chemotherapy drugs target in the cell cycle? And why?
S and M phases – kill rapidly replicating cells
Where do most cancers have mutations?
P53 regulator in the G1 phase due to damaged DNA
Every time cell divides they must…
replicate 6 billion basepairs
What is the accuracy and speed of DNA replication?
~100 nt/s
Define why DNA replication is described as semi-conservative
Each strand acts as a template
Two identical copies made: any mutations will be passed on
Where are nucleotides added to a primer strand during semi-conservative DNA replication?
3’ end
In which direction does DNA synthesis occur during semi-conservative DNA replication?
5’ to 3’ direction
Where is energy provided during semi-conservative DNA replication?
From the breakage of a triphosphate bond
What is formed between the new nucleotide and the primer strand during semi-conservative DNA replication?
Phosphodiester bond
Origins of replication forms….
Replication forks
Leading strand
continuous synthesis
Lagging strand
discontinuous synthesis
(Okazaki fragments)
Why is it called the lagging strand?
DNA is polar so the directions of the enzyme is moving in the 3’ to 5’ direction but synthesis only occurs in the 5’ to 3’ direction therefore it is synthesised in okazaki fragments
What occurs on the replication fork?
Joining of primer strands on the leading and lagging strand when DNA is unwinded
What does the sliding clamp do?
binds to DNA polymerase and tethers it to the DNA template preventing dissociation
What are the steps that occur during the replication fork in DNA synthesis?
ALL 8 STAGES
- DNA Helicase: Unwinds double helix
- DNA Primase: adds small RNA primer
- DNA Polymerase: binds, adds nucleotides to 5’ end (of leading strand)
- Exposed lagging strand protected by single-strand DNA binding proteins
- DNA Primase: adds small RNA primer
- DNA Polymerase: adds nucleotides to 3’ end (of lagging strand)
- DNA Nuclease: removes RNA primers, DNA polymerase fills in
- DNA Ligase: joins together small gaps
What is Werner Syndrome?
It is a Premature aging disorder
What causes Werner Syndrome in terms of DNA?
Mutation in a DNA Helicase (WRN)
Errors in DNA replication and DNA repair
What are the dangers of Werner syndrome? (4 things)
Increase in risk of cataracts, atherosclerosis, osteoporosis and cancer
What Prevents the accumulation of mutations?
Proof-reading capacity of DNA polymerase during DNA replication
Excision repair systems act throughout cell life repairing DNA damage
What do Excision repair systems do?
reduce error during DNA replication from:
1 mistake in 10^5 nt without proofreading
or repair to 1 mistake in 10^9 nt with both
Outline the steps of the excision repair system
- DNA nuclease cut
- DNA polymerase makes (adds) a new top strand using bottom strand as a template
- DNA ligase seals nick
What is Xeroderma pigmentosum?
mutation in UV repair
Unable to remove thymine dimers
Autosomal recessive disorder
What are the dangers of XP? (5 things)
Acute sun sensitivity
Hypo- and hyper-pigmentation
Multiple cancers at young age
Intellectual disability
Progressive degeneration
Mutation - Detailed definition
Any large/small change to DNA sequence (good or bad)
Assumes a deviation away from ‘normal’
Low frequency (<1% population)
Associated with disease (might refer to as gene variants when discussing with patients)
What is Single nucleotide polymorphism (SNP)?
Single base change in DNA sequence
Normal genetic variation in population
Synonymous
no change in amino acid sequence
Non-synonymous
change to amino acid sequence
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What is the difference between somatic and germline heritability?
Somatic - only effects body cells = not passed down
Germ-line - mutation in zygote affects all cells of the offspring
Name a Monogenic genetic diseases caused by substitution that losses a function
Sickle-cell anaemia
What type of mutation causes Cystic fibrosis (a loss of function)?
Deletion mutation
Huntington’s disease (a gain of function) is caused by?
Insertion mutation
What component is affected to cause sickle cell disease?
Single nucleotide substitution (A to T) in HBB gene (beta chain of haemoglobin)
What can sickle cells do?
Misshapen blood cells do not survive as long (can cause anaemia) and clog up capillaries
What is cystic fibrosis?
Abnormal lung mucus leading to infection
What is affected to cause cystic fibrosis?
3bp deletion in CFTR gene on chr 7 (cystic fibrosis transmembrane conductance regulator)
What is chr 7 and when it is damaged, what happens?
a chloride channel = changes the structure of this cannot transport to the surface of the cell
What is Hungtington’s disease? What does it do to a person?
Neurodegenerative disease
uncontrollable muscular movements
loss of memory and depression
difficulties with speech and swallowing
What is affected that causes Huntington’s disease?
Huntington’s genes change and become toxic and start to damage neurons in areas of the brain
Describe what happens to the huntington genes?
increase in the number of CAG trinucleotide repeats (encoding glutamine) in the Huntingtin (HTT) gene
What happens when CAG is repeated more than 40 times?
Polyglutamine residues stick together creating a toxic product (gain of function) which causes neuron cell death through multiple mechanisms
This causes enzymes to start looping back on themselves
What are the similarities between Gram-positive and Gram-negative?
Cytoplasmic membrane
Cell wall
Beta-Lactamase
Peptidoglycan
Penicillin-binding protein
What are the differences between Gram-positive and Gram-negative?
Outermembrane (beta-lactamase is here)
Has porins
Lipopolysaccharide
What does ATP-dependent chromatin remodeling complex do?
Decondenses chromatin
Slides nucleosomes along DNA, promote the exchange of histones or completely displace nucleosomes from DNA
How is genetic variation generated?
- Mutation
- Diploid cells
- Homologous recombination (HR) between chromosomes during meiosis
What is the difference between the line up of homologous chromosomes at metaphase plates mitosis and meiosis?
Mitosis - independent line up
Meiosis - Paired during independent assortment
What is the crossover point called ar homologous recombination?
Chiasma
What is non-disjunction?
When separation fails to occur causing either both sister chromatids or a homologous chromosome to be pulled to one pole of the cell
Trisomy usually fatal unless…
chromosome 21, 13 or 18
Which trisomy is known as Down’s syndrome?
21
Trisomy 13 is known as
Patau’s Syndrome
Trisomy 18 is known as
Edward’s Syndrome
Down syndrome (7 points)
1 in 750 live births
Non-specific effects
Learning difficulties
Broad flat face, slanting eyes
Congenital heart disease
Intestinal blockage
Enlarged colon
Give an example of a disease with monosomy
Turner Syndrome
Turner Syndrome (6 points)
Occurs in Females
Pubertal failure
Infertile
May have:
neck webbing, heart defects and horseshoe kidneys
What happens that causes Barr body?
X chromosome inactivation - this is Barr body
What is the genetic disease with XXY chromosomes?
Klinefelter syndrome (male, reduced fertility, lower IQ)
May go undiagnosed
What is the role of the Y chromosome?
SRY transcriptional regulator that causes testosterone production
Who is at more risk of non-disjunction?
Mature maternal parents - Important element of pre-natal testing
How are genetic diseases passed down through the generations?
Autosomal Dominant
Autosomal Recessive
X-linked
Give an example of an autosomal recessive disease
Cystic fibrosis
or sickle cell disease
On which gene is cystic fibrosis expressed?
CFTR gene on chr 7
On which gene is sickle cell disease expressed?
HBB gene on chr 11
Describe how sickle cell disease is expressed
Autosomal ‘Incomplete’ Recessive - small effect of mutation
Where is sickle cell disease most commonly found? Why?
African/Caribbean families
SCD carriers more resistance to malaria
How is SCD allele retained in the population?
Positive selection for HbS allele in malaria-endemic areas
Malaria parasites are unable to replicate inside heterozygote (carrier) red blood cells
Give an example of a disease caused by Autosomal dominant.
Huntington’s disease
On which gene is Huntington’s disease expressed?
HTT gene on chr 4
Give 2 examples of a X-linked recessive disease
Haemophilia A or Haemophilia
How is Haemophilia A expressed (gene terms)?
Deletions or inversions in F8 gene
Resulting in loss of function
Where is Haemophilia expressed?
F8 gene on X chr
What is Polygenic Diseases?
Combine gene variants associated with a specific disease
What genes does polymerase I make?
most rRNA genes
What genes does polymerase II make?
protein-coding genes
miRNA genes
plus genes for some small RNA’s (ones in spliceosomes)
most mRNAs
What genes does polymerase III make?
tRNA genes
5S rRNA gene
genes for many other small RNA
What is the negative connotation of RNA polymerase III?
Increases during cancer progression
What binds first before transcription?
Basal transcription factors
= 5’ end of the RNA has the correct sequence
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Give a Clinical examples of lack of transcription of a particular gene
Fragile-X syndrome
ATRX
What are the phenotypes of Fragile-X syndrome?
Large, protruding ears
Hyperextensible finger joints
Double-jointed thumbs
macro-orchidism (large testes)
learning difficulties
autism
How is Fragile-X syndrome expressed in DNA?
Expansion of CGG results in methylation of DNA in the promoter
This prevents the RNA polymerase complex assembling properly – no transcription of FMR1 gene’s protein
What is ATRX? (3)
ATRX is a protein that can unwind DNA.
It is part of large multiprotein complexes that control the local structure of chromatin
it is particularly associated with centromeres
What does ATRX (lack there of) cause? (2)
lack of ATRX shuts down transcription of a number of genes including alpha-globin
mutations in ATRX result in alpha-thalassemia (deficiency of alpha-globin) and mental retardation
Incorrect splicing can lead to which genetic disease?
β -thalassemia
What is Polyadenylation of the RNA?
Specific sequences encoded in the genome are transcribed into the RNA
Describe what happens in polyadenylation of the RNA
The RNA is cleaved and the polyA tail added to the 3’ end of the 5’ section
The result is an RNA which is capped, with its introns removed and a polyA tail
What allows very early recognition of infection of Gram negative bacteria?
Innate immune system is very sensitive to Lipopolysaccharide
What kind of receptors do LPS (Lipopolysaccharide) interact with?
Toll-Like Receptors (TLR) on:
Monocyte/macrophage lineage cells
and Vascular endothelium
What do LPS activate when they interact with receptors?
Inflammatory pathways
Coagulation and clotting pathways
Changes in endothelial integrity - make blood vessels leaky
TLR4 is a Toll-like receptor, what are the co-receptors for it?
CD14 and MD2
Why do we call endotoxins lipopolysaccharides?
other molecules like peptidoglycans can be referred to as endotoxins
How does Gram positive bacteria activate the immune response?
Lipoteichoic acid and Peptidoglycans
Using different Toll-like receptors
What is RNA polymerase bound with other factors and promotor called?
C-terminal domain
How does RNA polymerase starts making RNA?
C-terminal domain is phosphorylated
What do genes require to start transcription?
require a complex set of transcription factors (activator proteins) in place to bind to the RNA polymerase
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The C-terminal domain acts as an “assembly line” to co-ordinate the modifications of…
capping
splicing
polyadenylation
Describe the structure of a eukaryotic mRNA (left to right) - untranslated
5’ untranslated region (5’UTR)
Non-coding sequence
coding sequence
Non-coding sequence
poly-A tail : 3’ untranslated region (3’ UTR)
What does the 5’ cap (RNA capping do)
stabilises the RNA and also facilitates translation
What does polyadenylation do to eukaryotic mRNA?
Addition of poly-A tail which faciliates translation
What is unspliced RNA termed as?
pre-mRNA
How are introns removed from the primary transcript?
By a complex of RNAs and proteins called the spliceosome
How is accurate removal of introns achieved?
small nucleolar RNAs (snRNAs) within the spliceosome
What is β -thalassemia?
Clinical example of effects of incorrect RNA splicing
Anaemia from about 6 months of age
What causes β -thalassemia and what does it lead to?
aberrant processing – introns not spliced out properly
Results in premature stop codons
lack of protein
Describe how cystic fibrosis is caused due to incorrect splicing
exon 9 often skipped on mRNA (exon 8 to 10) –
loss of function
Mild form of cystic fibrosis
What is IGHD II? (2 things)
Familial isolated growth hormone deficiency type II = short stature
Clinical example of exon skipping
How is IGHD II caused?
Dominantly inherited disorder
Caused by mutations in the growth hormone gene (GH-1)
What is the use of introns?
Alternative splicing - 1 gene encodes for many different proteins due to different splicing variants
What do tRNA’s do?
interpret the mRNA codon and attach the correct amino-acid to the growing peptide chain
What allows the tRNA to link to the right amino acid?
tRNA synthase usinf ATP
What is the bond between the Amino acid and tRNA
high-energy bond
Describe the composition of ribosomes
mainly made of RNA with some accessory proteins
Has a large subunit (3 RNA) and a small subunit (1 RNA)
Summarise the stages of translation initiation
small ribosomal subunit scans along the RNA from the 5’ cap
carries the initiator tRNA and initiation factors
joined by large ribosomal subunit
polyribosome
A series of ribosomes can simultaneously translate protein for the same mRNA molecule (3’ and 5’ coiled next to each other)
What do antibiotics do to bacterial ribosomes?
Binding sites for antibiotics on the bacterial ribosome where tRNA usually goes to =
exploit structural and functional differences between the eukaryotic and prokaryotic ribosomes.
Name a genetic disease caused by a deleted frameshift mutation
congenital deafness
What happens to the gene in congenital deafness?
c35delG mutation causes ribosome to run into a stop codon
What is the name of the gene that causes congenital deafness?
connexin 26(GJB2) gene
What kind of genetic disease is congenital deafness?
autosomal recessive - must be homozygous
What kind of mutation causes sickle cell anaemia? And where?
mis-sense mutation in beta-globin protein
What codon was changed and what did it change to to cause sickle cell anaemia?
codon for glutamic acid changed to codon for valine
What is the effect of the mutated protein to red blood cells?
protein aggregates distort the red cells into a sickle shape
Name a genetic disease caused by a non-sense mutation
beta thalassemia
What causes beta thalassemia?
p.Gln39X (Q39X) mutation near the start of exon 2 which makes a stop codon
What happens during beta thalassemia?
mutated RNA is likely to undergo non-sense mediated decay - RNA degrades, less protein
What are microRNAs (miRNAs)? 3 points
21-22 nucleotides non-coding RNAs
Bind target sequences in 3’ UTR of mRNAs
Cause translational repression/degradation of mRNA transcripts
What were microRNA’s processed from?
longer precursors
What do precursor miRNA bind to after leaving the nucleus and entering the cytosol?
RISC proteins translate to make single-stranded miRNA and then search for complementary target mRNA
In the Regulation of gene expression by miRNAs, what happens when RISC finds extensive match?
RISC released and mRNA rapidly degraded
In the Regulation of gene expression by miRNAs, what happens when RISC finds less extensive match?
Translation reduced, mRNA sequestered and eventually degraded
What causes chronic lymphocytic leukaemia?
loss of microRNA expression
Explain how genes that are affected causes chronic lymphocytic leukaemia
13q-14.3 deletion
downregulation of miR-15a and miR-16-1
induces over-expression of BCL2
reduced apoptosis
Explain how miRNA-mediated regulates metastasis (The process by which cancer cells spread to other parts of the body)
miR-200 family can promote growth of the primary tumour and colonisation of metastases
miR-31 can inhibit movement and invasion of cancer cells to new location
How are microRNA expression profiles associated with various human cancers? Give examples
microRNA biomarker “signatures”
miR-21 is over-expressed in glioblastomas
miR-155 is over-expressed in various forms of B-cell malignancy
How do microRNAs have potential to be excellent biomarkers?
circulating microRNAs are found in circulating blood (serum and plasma) as well as other body fluids such as cerebrospinal fluid and urine
stable because they are protected inside exosomes/microvesicles and/or bound to proteins
How many miRNA’s are there in humans
> 2500 in humans
How can miRNAs be detected
using qRT-PCR on standard machines
List the different kinds of proteins
Structural proteins
Secreted structural proteins
Molecular motors
Intracellular proteins
Secreted proteins (like hormones)
Membrane bound proteins
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Structure of proteins
Amino group
Carboxyl group
R group (varient)
Hydrogen
How are Non-essential amino acids formed
Made by the body from the essential amino acids or by breakdown of proteins
How are essential amino acids formed
come from food in our diet
How are amino acids held together and what does this?
held together by peptide bonds generated by ribosomal peptidyl transferase
How are peptide bonds formed?
condensation reaction to form a covalent bond
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What determines how the protein folds (primary structure)
The distribution of hydrophobic and hydrophilic side chains along the backbone
Describe protein folding of primary structure
Hydrophobic chains tuck themselves inside the molecule to avoid water whilst the hydrophobic side chain stick outwards so they can form hydrogen bonds with the water and other polar molecules forming secondary structure
what are the most stable secondary structure?
alpha-helices and beta-pleated sheets
Sape of alpha-helix forms due to…
hydrogen bonds forming between carbonyl groups and an amino group 4aa along the chain
How many amino acids are there per turn in alpha-helix and where do the side chanins face?
3.6, face outwards
What are Molecular chaperones?
proteins that bind to the partially folded polypeptide chains and assist them in folding
Why are the class protein called heat shock proteins
their expression is increased at elevated temperatures, in response to exposure to stressful conditions.
What is the main amino acid that is mutated that causes cystic fibrosis?
70% of patients share mutation at aa508
Why is there Pulmonary obstruction in cystic fibrosis?
thickened mucus in the small airways causing recurrent bacterial infections
what happens to the amino acid and which one is it that causes CFTR?
deletion of the amino acid Phenylalanine at position 508
What does the mutant CTFR protein do to the body?
becomes stuck in the endoplasmic reticulum leading to reduced chloride conductance out of the cells
Alzheimer’s disease happens due to….
misfolding of amyloid beta causing amyloid beta plaques and tau causing intracellular neurofibrillary tangles (NFTs)
What do the misfolded proteins from Alzheimer’s disease do?
form tightly packed β-sheets that are very stable
How and what does Misfolded proteins cause?
The build-up of amyloid beta plaques and NFTs cause irreversible neuronal cell death via necroptosis
Where in the brain does neuronal cell death occur and over time what happens?
Initial damage is in the areas of the brain associated with memory formation
Over time the damage becomes more widespread leading to shrinkage of the brain
Where do Hydrophilic parts of the backbone form in alpha helices?
hydrogen bonds between carbonyl groups and amino groups
What do Hydrophobic side chains interact with?
hydrophobic hydrocarbon tails of the phospholipids
What is the hydrophilic backbone is shielded from?
the lipid environment of the membrane
How many amino acids are there in each beta strand?
5-8
How does the shape of the beta sheet formed?
hydrogen bonds forming between carbonyl groups and an amino group of a neighbouring chain
and side chains extend above and below β-sheet
Describe the structure of the secondary structure - turns
Turns are 3-5 amino acids long forming a sharp bend redirecting polypeptide backbone
Describe the structure of the secondary structure - loops
Loops have hydrophilic residues and are found on the surface of proteins
Describe the structure of the secondary structure - random coils
Polypeptide chains with random configuration
Where are many turns found?
in globular proteins that are tightly folded
What often connects beta sheets to alpha helices?
loops
What do secondary structures fold into?
Tertiary structure - three dimensional conformation
What are Barrels?
first and last strand form hydrogen bonds forming a barrel
What do beta barrels do in bacterial membrane/ proteins in human mitochondria?
allow ions to diffuse across membranes
How are Coiled Coil formed? Give an example of a coiled coil
2 or 3 α-helices wind around each other
keratin and collagen
What provides coiled coils strength?
Hydrophobic amino acids line up together where the helices meet.
How can tertiary structures be stabilised?
covalent cross linkages - disulfide bond between cysteines that are next to each other in the folded structure.
How are disulfide bonds formed?
They are formed by an enzyme in the endoplasmic reticulum.
Explain the composition of a large polypeptides
May fold into two or more domains, each with their own 3D shape, separated by short linker chains.
What parts of the protein are negatively charged?
Carboxylic group- glutamic and aspartic acid
What parts of the protein are positively charged?
N groups - Lysine, Arginine, Histidine
What will negatively charged DNA wrap around?
positively charged histones
What determines the shape?
The sequence of the amino acids - determines the 3D shape
Protein structures can form filaments, spheres, tubes, sheets etc
How can proteins be unfolded (denatured)?
Adding solvents (e.g. acids, bases) that disrupt the non-covalent interactions
The protein loses its secondary and tertiary structures.
How can proteins renature?
If the solvent is removed the protein will refold (renature) to the same shape as it started with.
What could denature proteins irreversibly and how?
Heat disrupts non-covalent interactions and provides enough energy to form new covalent bonds thus permanently altering the shape.
What allows proteins to go back to the same shape?
Protein folding funnel - Proteins spontaneously fold into a three-dimensional conformation of the lowest free energy
Why is the folding process energetically favourable?
It releases heat and increases the disorder in the universe
.
.
List the actions molecular chaperones can do:
do not change the 3D structure
speed up the folding process
prevent protein aggregation
reduce non-productive intermediates
Why are molecular chaperone proteins needed in the folding tunnel?
To help keep some proteins from misfolding on their way down the folding funnel, preventing them from straying into deep free energy wells (misfolded states).
How do molecular chaperone proteins stop proteins from misfolding?
Chaperones will bind the unfolded protein as it is made, shielding hydrophobic amino acids during the folding process.
What are Post translational modifications?
A processing event resulting from proteolytic cleavage or the covalent addition of a modifying group.
What is proteolytic cleavage?
the process of breaking the peptide bonds between amino acids in proteins
.
.
Ubiquitination
Addition of ubiquitin can target the protein for destruction by the proteasome
Glycosylation
Addition of carbohydrates to specific sites on the protein
Phosphorylation
Addition of a phosphate to specific amino acidsregulating the activity of the protein
What does N-linked glycosylationrequire?
Participation of a special lipid called dolichol phosphate
What is the proteasome?
a protein complex that degrades proteins by proteolysis a chemical reaction that breaks peptide bonds
How does protein structure relate to medicine?
Some mutations in a gene encoding a protein will not affect the aa (amino acid) sequence
Other mutations cause a change in the aa sequence which may cause the tertiary structure to become altered affecting the function - aggregation
What is the peptide backbone?
the N-terminus and the C-terminus that repeats -N-C-C-
What stabilises the flexible peptide backbone?
by weak non-covalent bonds (Hydrogen bonds), electrostatic reactions (between charged groups) and van der Waals forces (when molecules (atoms) are very close to each other (due to fluctuating electrical charge)).
They are all weak bonds but when many occur simultaneously that can hold the structure together.
What is Cystic Fibrosis a inherited disease of?
The secretory glands
What causes Cystic fibrosis?
DNA mutation on CFTR gene on chr 7 that alters protein shape
How many mutations are detected and
70% of patients share mutation at in cystic fibrosis?
800
amino acid 508
What is the mutation that causes cystic fibrosis?
deletion of the amino acid Phenylalanine at position 508
CTT gets deleted
Why does Cystic fibrosis cause Pulmonary obstruction?
Due to thickened mucus in the small airways causing recurrent bacterial infections
What leads to the reduced chloride conductance out of the cells in cystic fibrosis?
Mutant CTFR protein becomes stuck in the endoplasmic reticulum
What is Lumacaftor?
Small moleculedrugs that can bind to and stabilise the protein of the Phe508 deletion gene mutation, meaning more CFTR can be trafficked to the cell surface
What is the percentage of AD accounts for
dementia diagnoses
60 to 70%
Even though AD (Alzheimer’s) is sporadic what causes it?
a combination of genetic, lifestyle and environmental factors, with <1% of cases classed as familial AD, which is inherited
What causes Alzheimer’s disease ?
due to the misfolding of amyloid beta causing amyloid beta plaques and tau causing intracellular neurofibrillary tangles (NFTs)
Wat do the misfolded proteins form in AD?
The misfolded proteins form tightly packed β-sheets that are very stable
What is the major genetic risk factor of AD?
carrying the apoE4 allele of the apolipoprotein E (APOE) gene involved in cholesterol transport.
How is AD characterised?
progressive memory loss and cognitive decline
What happens to proteins synthesized by ribosomes in the cytosol?
Either remain in the cytosol or get transported into mitochondria, peroxisomes or the nucleus
What happens to proteins synthesized by ribosomes at the Endoplasmic Reticulum (ER)?
Destined for secretion, the plasma membrane, secretory vesicles and lysosomes then sorted in the Golgi apparatus
What happens after the protein leaves the ribosome?
Part of the amino acid sequence may contain a signal sequence (or sorting sequence) - It provides the information as to the correct location of that protein
Where do proteins without a signal sequence?
Remain in the cytosol
What proteins normally remain folded?
Proteins that are transported through pores or by vesicles
What proteins must be unfolded?
Proteins that are transported across or into membranes
.
.
What do nuclear pores consist of?
complex of around 30 different proteins termed nucleoporins that coat the pore lumen
What mediates the transport of nuclear proteins into the nucleus?
nuclear localisation signal (NLS)
What recognises nuclear localisation signal (NLS) and what does this cause?
recognized by importin (also called nuclear import receptor) which then binds to proteins within the cytosolic fibrils
What does Importin do and how does it perform its function?
Importin carries the protein into the nucleus by disrupting the gel-like mesh of the proteins lining the channel
What is the interaction inside the nucleus when the protein is carried into the nucleus?
interaction with Ran causes release of the protein and returns importin back to the cytosolic side of the pore ready to start again
What happens to mitochondria proteins during import?
They are unfolded
What recognises the signal sequence of a mitochondrial protein?
an import receptor on outer membrane of the mitochondria
What passes the protein through the mitochondrial membranes?
Protein translocators
Which area does the import receptor and protein translocator diffuse to? (mitochondrial proteins)
where membranes come into close proximity to engage the second translocator
What happens when the mitochondrial protein is inside the mitochondrial matrix?
the protein folds and the signal peptide is cleaved off
What directs ribosomes to ER? How does it?
signal recognition particle (SRP) binds to the signal sequence and the ribosome
What happens after (SRP) binds to the signal sequence and the ribosome?
It attaches to the signal sequence and ribosome complex and this binds to a receptor on the ER membrane at which point SRP is released, then the ribosome passes to the protein translocator which binds the signal sequence
How does Protein initially import into the ER?
Via The protein translocator binds the ER signal sequence during translation and threads the polypeptide across the lipid bilayer as a loop.
What happens after when the ER signal sequence binds to the protein translocator?
signal peptide is removed from the growing polypeptide by a signal peptidase and left in the membrane to be degraded
What happens as the polypeptide crosses the ER membrane?
the molecular chaperone BiP binds ready to help fold the protein within the ER
What happens Once protein synthesis is complete in ER?
the polypeptide is released as a soluble protein in the ER lumen and the protein translocator closes
Describe the Insertion of a single-pass transmembrane protein in the membrane
An N-terminal ER signal sequence initiates the transfer, which is then halted by a stop-transfer sequence.
Once inserted the N-terminal signal sequence is removed by a signal peptidase.
Describe the Insertion of a double-pass transmembrane protein in the membrane
A double-pass transmembrane protein has an internal ER signal sequence.
An internal sequence acts as a start-transfer signal and also anchors the protein in the membrane.
When a stop-transfer sequence enters the protein translocator, the translocator discharges both sequences into the lipid bilayer.
What is Glycosylation during protein modification?
The addition of polysaccharide done by oligosaccharide Transferase with active site exposed in lumen
.
.
When does glycosylation occur?
When an appropriate asparagine (Asn) enters the ER lumen, it is glycosylated by the covalent addition of a branched oligosaccharide side chain.
Why is glycosylation important? What does it do?
Glycosylation is critical for the physiological and pathological cellular functions of many proteins.
Changes in glycosylation can modulate inflammatory responses, enable viral immune escape, promote cancer cell metastasis, or regulate apoptosis
What happens to Misfolded proteins in the ER?
retained by chaperones until they can fold correctly or exported into the cytosol for degradation by the proteasome
What happens when there is An excess of unfolded proteins in the ER?
triggers the unfolded protein response (UPR)
Describe the unfolded protein response (UPR)
Sensor proteins become activated by the accumulation of protein and then stimulate an expansion of the ER, increase number of chaperones and reduce the amount of new proteins entering the ER
What happens to Proteins transported from the ER?
Go through the Golgi apparatus to the plasma membrane or via early and late endosomes to lysosomes.
What happens to Endocytosed vesicles derived from the plasma membrane?
Delivered to early endosomes and usually on to lysosomes via the late endosome.
What is the Constitutive pathway?
Continual secretion of soluble proteins from the cell.
= unregulated exocytosis
This route also replaces proteins and lipids in the membrane.
They form newly synthesised: soluble proteins, plasma membrane lipids and plasma membrane protein
What is the regulated pathway?
Secretory vesicles store proteins until an extracellular signal stimulates their secretion.
= regulated exocytosis - requires signal transduction from extracellular signal (hormone / neurotransmitter)
How do Vesicles need to maintain their identity to ensure the correct proteins are transported and that they reach the correct location?
Coat proteins
Vesicles formed at membranes have distinct proteins on the cytosolic side which aid in vesicle budding.
Coat proteins shape the membrane into a bud and directly or indirectly capture the cargo proteins for transport.
What are the types of coated vesicle?
Clathrin, COPII or COPI coated
What are the types of coat proteins?
clathrin + (adaptin 1 or 2), COPII , COPI
Where are Clathrin-coated vesicles formed?
form from the cell membrane and from Golgi apparatus
What are Clathrin molecules made up of?
three chains that form basket-like cages
.
.
When is clathrin involved?
only involved in shaping the budding membrane
What do adaptins do?
select the cargo molecules
What do Cargo receptors bound to molecules interact with? Then what happens
adaptin which in turn interacts with clathrin, it goes from a bud formation to a vesicle formation
When a clathrin-coated vesicle is formed, what allows it to still be attached to the extracelluar space?
Dynamin (a GTP binding protein) assembles around the neck of budding vesicles to pinch off the vesicle.
What happens when the clathrin-coated vesicle detaches from the extracellular space?
After budding the coat proteins are removed and the naked vesicle can fuse with its target membrane.
What helps direct transport vesicles to their target membranes?
Rab proteins, tethering proteins, and SNAREs
Describe the process of Recognition of vesicles with a cargo protein at target membrane
tethering and docking
- A tethering protein binds to Rab on the vesicle. (tethering)
- The vesicle docks on its correct target membrane. (docking)
- A v-SNARE on the vesicle binds to a complementary t-SNARE (on the target membrane.
Describe the process of Vesicle docking and membrane fusion of a transport vesicle
- v-SNAREs and t-SNAREs bring the lipid bilayers close together.
- They wind together squeezing water molecules away from the membranes, allowing their lipids to flow together to form a continuous bilayer.
- After fusion, the SNAREs are pried apart so that they can be used again.
How can Viruses can enter cells via membrane fusion?
After recognizing specific proteins (in this case CD4) viruses can fuse their membrane with the plasma membrane of the target cell.
Viral nucleic acids enter the cell and replicate.
How can Viruses can enter cells via receptor-mediated endocytosis?
Viruses taken up by receptor-mediated endocytosis will fuse with lysosomes, where the low pH releases the viral genome into the cytoplasm.
Describe the 3 fates of receptor proteins following their endocytosis depending on the type of receptor during receptor translocation
Recycling
Receptors are returned to the same plasma membrane domain from which they came.
Transcytosis
Receptors are returned to a different area of the plasma membrane.
Degradation
Receptors are delivered to lysosomes to be degraded.
Describe the components in Lysosomes or late endosomes
They have a lower pH and contain acid-dependent hydrolytic enzymes.
What is Endocytosis?
is the uptake of material from outside the cell often induced by receptor binding.
What is autophagy?
Autophagy is the breakdown of the cells own proteins and organelles by enclosing them in a membrane that fuses with the lysosome
Vesicles can be destined for lysosomal degradation, what are the 3 ways it can do so?
Phagocytosis, Endocytosis, Autophagy
What is the difference between intercellular and Intracellular signaling?
Intercellular signalling – between cells (extracellular signalling)
Intracellular signalling – inside cells
Why do cells need to communicate?
A cell must coordinate its behaviour:
Communication with neighbouring cells
Adaption of metabolism
Movement, e.g. muscle contraction
To induce/decease Growth (division) if the need arises
Respond to danger signals
When new cells are needed, stem cells must:
Upregulate proliferation by the right amount
and Differentiation to the desired cell type
Types of molecules used as intercellular signals
Proteins e.g. Interferon, insulin
Peptides e.g. glucagon, growth hormone, produced by cleavage of proteins
Small chemicals: steroids, made from cholesterol e.g. cortisol
or amino acid metabolites e.g. adrenaline
Dissolved gases e.g. Nitric oxide
What is the ligand?
The signalling molecule that binds to a receptor
What are the conditions for a cell to respond to a signal?
the ligand must be present
the responding cell must possess the corresponding receptor
the receptors must be correctly coupled to an intracellular signaling pathway
What does Gene expression during differentiation determine?
Determines which receptors are synthesised by a cell and the level of expression
What does Ligand binding do?
changes the activities of the intracellular domains of the receptor, which initiates the response
What are the Three main categories of membrane receptors?
Receptors linked to ion channels
G protein-coupled receptors (GPCRs)
= Receptors linked to G proteins
Receptors linked to enzymes
Describe the fast effect of receptor activation
Intracellular signaling pathway -> altered protein function -> altered cytoplasmic machinery -> altered cell behavior
Describe the slow effect of receptor activation
Alter protein synthesis -> altered cytoplasmic -> altered cell behaviour
What can bind to the Receptors that can be located inside the cell?
Dissolved gas (e.g. Nitric oxide) and hydrophobic molecules (e.g. steroid hormone cortisol) cross the cell membrane and directly bind to receptors in the cytosol or the nucleus
What are the 2 contradicting actions adrenaline can do?
Causes contraction of the smooth muscle cells in the blood vessels supplying the gut
but
adrenaline also causes relaxation of the smooth muscle cells in the blood vessels supplying the skeletal muscles
What is the alpha action of the adrenal response?
contraction of smooth muscle in the gut blood vessels
What is the beta action of the adrenal response?
relaxation of smooth muscle in skeletal muscle blood vessels
What is the receptor type of adrenaline?
GPCR - G protein-coupled receptors
name a Muscarinic-type receptor
G protein couple receptor (GPCR)
name a Nicotinic-type receptor
Na+/K+ channel
What activates salivary gland cells to secrete?
M1 receptor signals via Gq (Gq alpha subunit), PLC (phospholipase C), IP3
What activates heart pacemaker cells to decrease the rate of firing?
M2 receptor Gi inhibits adenylyl cyclase decreasing cAMP
Which signal is long range?
Endocrine signals
Which signal is short range?
Paracrine signals
Neuronal signals
Autocrine signals
Juxtacrine signals
What happens to Hormones are produced by an endocrine gland?
released into the bloodstream where they act on target cells at a distance locations
Hormones circulating in the blood come into contact with most cells within the body
What is the difference between type 1 and 2 diabetes?
In Type 1 diabetes the signal is missing.
In Type 2 diabetes the target cells do not respond adequately to the signal.
What is Cortisol?
Steroid hormone made from cholesterol that is made by the adrenal gland on the kidneys that regulates metabolism of proteins and body fat
What does cortisol bind to and what does it cause?
The glucocorticoid receptor (GR) activates and suppresses gene expression producing both metabolic and anti-inflammatory effects.
What can an increase in cortisol cause?
Increased cortisol causes suppression of the immune system and can cause an increase in blood glucose levels by releasing glucose from cells (glucogenesis).
What are the 3 Synthetic hormones that activate the Glucocorticoid receptor?
Hydrocortisone (cortisol)
Prednisolone
Dexamethasone
What are the diseases that can be treated with steroids?
Autoimmunity e.g. Psoriasis, ulcerative colitis
Allergic reactions e.g. Urticaria (hives)
Inflammatory conditions e.g. Asthma
What are Paracrine signals?
Signalling molecules (local mediators) are released from one cell and diffuse locally to neighbouring cells
Give 2 examples of protein Paracrine signals
Cytokines generate an immune response
Platelet-derived growth factor stimulates cell proliferation
What is the name of an amino acid derivative paracrine signal?
Histamine promotes local inflammation
What is the name of a dissolved gas paracrine signal?
Nitric oxide relaxes smooth muscle, dilates blood vessels
How is Nitric oxide produced?
produced from arg when blood vessel wall endothelial cells are stimulated by acetylcholine
Describe what Nitric Oxide does
diffuses into the blood vessel wall and activates its target enzyme guanylate cyclase which catalyzes the conversion of GTP to cGMP which causes rapid relaxation of smooth muscle
What does Cyclic GMP cause?
the smooth muscle cells to relax, and thus increases blood flow through the blood vessel
Why is the effect of nitric oxide The effect is short-lived?
as the cyclic GMP is quickly hydrolysed to GMP by phosphodiesterase (half-life ~10 seconds)
What is the clinical use of nitric oxide?
The pain from angina can be relieved very rapidly by administering nitroglycerin which is converted in the body to NO, improving blood flow in the coronary arteries.
What causes angina?
caused by inadequate blood flow to the heart muscles
How is nitroglycerin (taken as a pill/patch) converted to nitric oxide?
It is converted to NO by mitochondrial aldehyde dehydrogenase (ALDH2).
Name and explain 4 Neurotransmitters
Adrenaline
= Regulates attentiveness and mental focus
As a hormone (released by the adrenal gland) adrenaline redirects blood to the muscles and increases the conversion of glycogen to glucose
Acetylcholine
= Released by motor neurons innervating muscle cells
Serotonin
= Important in the modulation of mood levels are modulated by antidepressants
Dopamine
= Important for the fine-tuning of motion low levels observed in Parkinson’s disease
What is Autocrine signals?
Cells secrete signalling molecules that bind their own receptors to generate a change in their own behaviour.
What 2 types of signaling causes activation of the cells within a tumour?
Growth factors released by cancer cells can activate the cells within a tumour by autocrine and paracrine signalling, stimulating cell proliferation
What is juxtacrine signalling?
Contact-dependent signalling: immediate neighbours signal to each other via membrane-bound molecules.
What are gap junctions?
channels that form between cells allowing diffusion of small molecules such as ions, nucleotides and sugars
What is contact-dependent receptor ligand binding?
Direct cell to cell communication or interactions with the extracellular matrix (ECM)
What is the purpose of gap junctions?
Gap junctions provide neighbouring cells with a direct communication link that can be opened or closed in response to the cell environment
How are gap junctions formed?
formed by channels called connexons
What are connexons?
connexons consist of 6 protein subunits
There are 20 types of subunit exist
Connexons can be built from the same or different subunits
Signalling cascades phosphorylate the connexions to allow opening and closing of the gap junctions.
What is the purpose of Gap junctions between heart muscle cells?
allow waves of electrical excitation to pass quickly through the tissue
Where in the body can gap junctions appear? And why?
appear in the myometrium of the uterus towards to the end of pregnancy, where they help coordinate uterine contractions during childbirth.
What does Contact dependent receptor ligand binding require?
Requires direct contact between the
T cell receptor – Antigen presenting cell
What are the hallmarks of cancer? (6 original) - features of cancer
Sustaining proliferative signaling
Evading growth suppressors
Activating invasion and metastasis
Enabling replicative immortality
Inducing angiogenesis
Resisting cell death
What are the newer hallmarks of cancer?
Emerging hallmarks:
avoiding immune destruction
Deregulating cellular energetics
Enabling characteristics:
Genome instability and mutation
Tumour-promoting inflammation
Describe what Sustaining proliferative signaling means/does
external growth factors bind to receptor on cell
Causes an intracellular signalling pathway (cascade)
Here ras protein has been mutated
this causes Promotes tumour growth
What is the most common growth suppressor that is evaded in the development of cancer?
P53 – a pivotal molecule in sensing dna strand has been distorted, allows the cell to pause, go to arrest, tumour suppressed
What are the 6 ways that cancer cells exhibit ways of inhibiting apoptosis?
Main: Bcl-2, Bcl-xL overexpression
Caspase-3,-8,-9 activation (cascade) inhibited
Loss of p53 function
TNFAIPS increases
MAPK activity decreases
Deregulated NF-kB
Leads to the survival and drug resistance of cancer cells
Describe Limitless replicative capacity
that leads to enabling replicative immortality
Telomeres are chromosomes ends made by telomerase early in development then normally turned off but in cancer cells, telomeres shorten with each cell division, and telomeres shorten too much but still continues to divide and senescence is bypassed and there isreactivation or up-regulation of telomerase = leads to immortal cancer cells
What is tumour angiogenesis?
Tumour secretes VEGF which increases blood vessel expression and movement to tumour and now it has increased blood supply
Describe primary breast carcinoma
- Primary metastic tumour in breast
- proliferation/angiogenesis (of blood vessel)
- Detachment/invasion into lymphatics, venules and capillaries
- Embolism/circulation
interacts with platelets, lymphocytes and other blood components - Transports to the heart which causes arrest in organs such as lungs
- cancer cells adhere to vessel walls
- This causes extravasation (Leakage of a fluid)
- Prolifeation/angiogenesis outside vessel wall then metastasis in lungs
Describe the dose-response relationship
A sigmoidal (S-shaped)
dose (drug conc in microM) -response curve
What are Agonist?
a chemical that binds to and activates a receptor to produce a biological response
What are Antagonist?
Blocks the actions of an agonist.
A true (silent) antagonist does not produce any biological response on its own
What are the Factors affecting Drug Responses?
AGE - GENETICS – ENZYME ACTIVITY - BODY COMPOSITION – PREVIOUS EXPOSURE
Describe the Drug-Receptor Interaction
Drug + receptor (lock and key) -> (equilibrium between Ka {association constant} and Kd {dissociation constant} ) = Drug-receptor complex -> (intrisic activity) = Response
What is the formula for affinity?
Ka {association constant} / Kd {dissociation constant}
What are Competitive Antagonist?
Antagonist competes with agonist
for the same receptor, e.g. atropine at acetylcholine receptors
What are Non-competitive Antagonists?
Antagonist binds irreversibly with the receptor – an irreversible antagonist - no agonist can bind
OR antagonist interacts with a different part of the receptor (not the agonist binding site) and inactivates it - Also called an allosteric effect or an allosteric antagonist
How does Competitive Antagonism work?
Increasing concentrations of the agonist eventually out-competes the antagonist to produce a biological effect
Maximum response is obtained
What are Constitutively-active Receptors
? (can you give examples)
Receptors that have a background activity even when an agonist is not present
e.g. histamine receptors, some GABA receptors
What forms the basis of the Two-State Model of Receptors?
Receptors can exits in two states: Resting (R) and Active (R*)
There is an equilibrium between the resting and active states
Under baseline circumstances, most receptors remain in the resting state
For “Constitutively Active” receptors, What is the Two-State Model of Receptors?
slightly more receptors are in the Active (R*) state and this generates a background response without an agonist present
Why can agonists generate a response from the two-state model of receptors?
Agonists have a higher affinity for the receptor in its active state.
When R* is activated by an agonist, an increased response is obtained.
The equilibrium below moves towards the active state R*
What are INVERSE AGONISTS?
agonists which interact with the same receptor produce an opposite response because:
They have an increased affinity for the receptor in its resting state R
Describe what inverse antagonists can do and it’s a condition
Inverse agonists produce an opposite response
By reducing the constitutive background activity of receptors which are in their active state
Inverse agonists will only produce a response on receptors that have
constitutive receptor activity by reducing their background response
What is the effect of
an Antagonist on receptors in the resting and active state?
A neutral antagonist has an equal affinity for both the resting state R and the active state R* of the receptor
What are the Targets for Drug Action?
- Ion channels
- Enzymes
- Carrier Molecules
- Receptors
What are the receptors that are targets for drug action?
- Type 1: Ligand-gated ion channels
- Type 2: G-protein-coupled receptors
- Type 3: Enzyme-linked and related receptors (also known as Kinase-linked receptors)
- Type 4: Nuclear “intracellular” receptors
Describe how drugs target ion channels, can you give an example
Some drugs can interact DIRECTLY with ion channels
e.g. lidocaine (lignocaine), can physically block Na+ channels in nerves
= local anesthetic effect
What mediates pain and inflammation?
The enzyme cyclo-oxygeanse (COX) converts arachidonic acid into prostaglandins (PGs)
What does aspirin do?
The drug anti-inflammatory drug aspirin (acetyl-salicylic acid) specifically targets COX and inhibits its activity
What is the general effect of aspirin?
to reduce the generation of inflammatory prostaglandins which results in pain relief (less pain)
What controls the composition of urine in the kidneys?
The Na+K+2Cl- pump
What drug increases urine production?
The “loop-diuretic” drug (water tablets) furosemide
Describe the diuretic effect
More Na+ excreted
More H2O excreted
What is the pharmacological effect of the loop-diuretic drug?
REDUCED OEDEMA = (water retention)
Give an example of a carrier/transporter target for drug action (specifically in the kidneys)
Receptor for Furosemide - ion transporter for Na+, K+ and 2Cl-
What are Type 1 receptors?
Ligand-gated ion channels - Receptors on the ion channel
Ions cannot dissolve into the fatty layer - they need to have an ion channel that will allow them to move into and out of the channel
What is GABA?
GABA (gamma-aminobutyric acid)
The predominant inhibitory neurotransmitter in brain tissue
What causes brain tissues to remain sedated until excited?
GABA interacts with GABA receptor (receptor gates Cl- channels)
Allows Cl- to enter the cell
Makes depolarisation difficult
Stabilises tissue = Produces sedation
What are Type 2 receptors?
G-Protein-coupled Receptors
G-protein coupled receptors are involved in cAMP pathway, describe its involvement
Signalling molecule (Endogenous ligand or drug e.g. noradrenaline, adrenaline) binds to G-protein coupled receptors (Gs or Gi) using GTP
Triggers Adenylate Cyclase (AC)
Catalyses cAMP production (second messenger) from ATP and broken down by phosphodiesterase and binds to the target protein = protein kinase A
= PHYSIOLOGICAL
RESPONSE
What is the difference between Gs and Gi?
Gs activation increases AC activity
and increases cAMP production
Gi activation reduces AC activity
and decreases cAMP production
What does cAMP stand for?
cyclic 3’,5’-adenosine monophosphate
G-protein coupled receptors are involved in the calcium pathway, describe its involvement
Signal molecule (Ligand or agonist) e.g. histamine binds to receptor coupled with G protein Gq (G alpha subunit) with GTP
Gq can freely activate PLC
Triggers Phospho-lipase C (PLC) which catalyses the formation of IP3 or DAG (second messenger) from PIP2
This increases cytosolic Ca2+ released from the intracellular stores
This affects a target protein that causes a physiological response
What does IP3 stand for?
Inositol 1,4,5-trisphosphate
What does PIP2 stand for?
phosphatidylinositol 4,5-bisphosphate
What do Physiological beta-2 receptor agonists take and which pathway does it take?
Noradrenaline (also adrenaline) binds to beta-2 receptor
Pathway = cAMP
What is the target protein and physiological response of Physiological beta-2 receptor agonists?
Smooth muscle relaxation
widening the airways (bronchi) = Bronchodilation
What breaks down cAMP when Physiological beta-2 receptor agonists, bind to beta-2 receptors coupled with G protein?
Broken down by phosphodiesterase
Explain the physiological differences of when someone has asthma compared to someone who doesn’t
Less Adrenaline
Beta-2 receptor is less effective
cAMP concentration is lowered
Less smooth muscle relaxation
Bronchoconstriction
Name a Synthetic beta-2 receptor agonist
Salbutamol
Why can’t some people do not respond to Synthetic beta-2 receptor agonists?
Due to genetic differences in the
molecular structure of their β2 receptors
What is a Type 3 receptor?
Enzyme-coupled receptors
Describe the action of Enzyme-linked receptors (quick = minutes)
Enzyme-linked type-3 receptor binds to growth factor
It activates a relay protein and activates so on… which causes a Protein phosphorylation cascade
Causes direct cellular effects
Describe the action of Enzyme-linked receptors (slow = hours)
Enzyme-linked type-3 receptor binds to growth factor
It activates a relay protein and activates so on… which causes a Protein phosphorylation cascade
Causes gene transcription and protein synthesis
Causes cellular effects (Modify protein production)
What are Type 4 receptors?
Nuclear Receptors: Intracellular Receptors
Also known as steroid hormone receptors
What is the action of nuclear receptors?
They are normally at rest in the cytosol (inactive)
Move to the nucleus when they bind their activating ligand – lipid soluble does not need channel
They Control gene expression and lead to protein synthesis
What are the Physiological Responses to Cellular Signals?
Activation of enzyme activity
Change in cytoskeletal organisation
Change in ion permeability
Activation of DNA synthesis
Activation of RNA synthesis
What are the receptors with several molecular varieties or “subtypes”
beta-receptors (beta1, 2, 3) (beta1 = heart rate, tremor, beta2 = bronchodilation…)
5-hydroxytryptamine (5-HT) receptors
= 13 cloned subtypes so far
Distinct subtypes of acetylcholine receptors (AChR) are located in in different parts of the brain and differ from AChRs in muscle
Beta receptors are also located on the heart where noradrenaline (and adrenaline) act as agonists to…
increase force of contraction
In high blood pressure, a drug called …
atenolol blocks beta-1 receptors to reduce blood pressure: an antagonist effect by a “beta-blocker”
What is Receptor Desensitisation?
a short term effect
happens very quickly (days)
involves a loss of intrinsic activity of receptor complexes
What is a Receptor Down-Regulation?
a longer term effect (weeks)
involves a loss of number of receptors from cell surface
takes longer to recover
What are the Other Mechanisms for a Loss of Effect of a Drug? And examples
Exhaustion of mediators
e.g. constant drug activation causes one or more mediators to “run out”
Increased metabolic degradation
e.g. by induction of drug metabolising enzymes e.g. CYP450 leading to increased breakdown of drug
Physiological adaptation
e.g. by production of new cells which lack active receptors or induction of resistance genes
Active extrusion of a drug from a cell
e.g. Multi-Drug Resistance = MDR - especially relevant for anticancer and antibiotic drugs: a major cause of antibiotic resistance
What are the 4 Effects of Drug Action?
Therapeutic Effects:
Drug produces the intended biological effect
Side Effects:
Nuisance = e.g. dry mouth
Or Can become harmful, e.g. sedation
Adverse Effects:
Undesired effects that can be harmful, e.g. allergies, anaphylaxis, anaphylactic shock
Toxic Effects: produces a metabolite that causes =
Drug poisoning, harmful, may be life-threatening
e.g. liver damage associated with paracetamol overdose
When and why could unwanted effects occur?
can occur when a drug interacts at the same type of receptor but at different sites around the body
may also occur if a drug is able to interact with more than one type (e.g. subtype) of receptor
Why are antagonists more likely to produce unwanted effects?
Antagonists are more likely to interact with multiple receptor types because there are fewer constraints for molecular structure
What are the benefits of observing unwanted effects from drug interactions?
Many unwanted effects can be predicted from a knowledge of the drug actions
‘Side-effects’ are often used to advantage in the treatment of other conditions,
e.g. constipation caused by opioid agonists can be anti-diarrhoreal
What are the 2 parts of the autonomic nervouse system?
Parasympathetic and Sympathetic
Parasympathetic activation causes…
contraction
constriction
secretion
The “Rest and Digest” system
Sympathetic activation causes…
relaxation
dilation
= More blood into your lungs and muscles
secretion
The “Fight or Flight” system
Describe briefly the action of neurotransmitters in the parasympathetic system
Nicotinic receptors releases acetylcholine and bind to -> Muscarinic Receptors which releases acetylcholine
Describe briefly the action of neurotransmitters in the Sympathetic system
Nicotinic receptors releases acetylcholine and binds to -> Adrenoceptors releasing noradrenaline
What does the autonomic nervous system control in hypertension?
Both cardiac output and peripheral resistance (The resistance to blood flow resulting from the friction of blood against the walls of vessels)
What is classified as High BP?
> 130/90 mmHg
or > 150/90 mmHg if over 80 years old
Drugs can act to lower blood pressure by:
(1) Decreasing cardiac output
(2) Decreasing peripheral resistance
How is cardiac output decreased?
by increasing parasympathetic nervous system activity
What does the treatment of hypertension targets?
The heart in the Parasympathetic system and decreases rate and force of contraction
How could we decrease rate and force of contraction in the parasympathetic system to treat hypertension?
(1) Give a muscarinic agonist, e.g. acetylcholine, carbachol or pilocarpine
(2) Prevent breakdown of acetylcholine. Use an anticholinesterase aka acetylcholinesterase inhibitor e.g. neostigmine
Would treatment of hypertension using drugs work?
bymuscarinic agonst or prevent breakdown of acetylcholine
Yes - decreased cardiac output
But no parasympathetic innervation of blood vessels, so no effect of peripheral resistance
So overall a modest effect on BP
What are the Side Effects of using drugs to treat hypertension?
Stimulation of the parasympathetic NS:
Contraction of ciliary muscles and iris
Secretion of lacrimal and salivary glands
Visual disturbances
Lacrimation & salivation
Bronchoconstriction in the lungs
Stomach cramps - contraction of stomach
Constipation - Contraction of small intestine
Incontinence – contraction of the bladder
How could we decrease blood pressure?
decreasing sympathetic nervous system activity
How could we use drugs to decrease blood pressure?
Give an adrenoceptor antagonist
Prevent release of noradrenaline
Does using drugs to decrease blood pressure work?
Yes - Decreased cardiac output
Sympathetic innervation of blood vessels: block this to reduce peripheral resistance
Noradrenaline acts on two subtypes of adrenoceptors:
Alpha and Beta adrenoceptors
What does Beta(1)-adrenoceptors cause?
inotropism and chronotropism
(increased force of contraction of the heart and increased heart rate)
What does Alpha-adrenoceptors cause?
vasoconstriction
What does Beta(2)-adrenoceptors cause?
vasodilation
What does using Mixed alpha/beta antagonist,
e.g. carvedilol, labetalol cause ?
Decreased cardiac output
Decreased blood flow to skeletal muscle
Increased blood flow to skin & gut
What are the side effects of using alpha/beta antagonists to decrease blood pressure?
Bronchoconstriction – inhibits opening of airways
Postural hypotension
Bradycardia
Muscle weakness/fatigue
Cold extremities
What does Selective alpha antagonist,
e.g. phentolamine
do?
Increased blood flow to skin & gut (decreased peripheral resistance)
BUT No direct effect on heart
Decreased blood pressure
What are the side effects of using Selective alpha antagonist,
e.g. phentolamine
?
Headache
Postural hypotension
Reflex tachycardia – it thinks blood pressure too low
What does using Nonselective beta antagonist, e.g. propranolol do?
Decreased cardiac output and blood pressure
But Decreased blood flow to skeletal muscle due to increased peripheral resistance
What are the side effects of using Nonselective beta
antagonist, e.g. propanolol?
Bronchoconstriction
Muscle weakness / fatigue
Using drugs what do we want to target to decrease blood pressure?
Beta(1) receptors on heart
Avoid Beta(2) receptors and in airways and lungs and also on blood vessels
What does Selective beta(1) antagonist,
e.g. atenolol cause?
Decreased cardiac output
No effect blood flow to skeletal muscle
Decreased blood pressure
No bronchoconstriction (depends on dose)
What happens when using a combined beta(1) antagonist & beta(2) agonist?
Decreased cardiac output
Bronchodilation
Increased blood flow to skeletal muscle (decreased peripheral resistance)
Decreased blood pressure:
No bronchoconstriction
No muscle weakness / fatigue
What is the disadvantage of using a combined beta(1) antagonist & beta(2) agonist?
Experimental /
in development
If we could decrease blood flow to the nose, we…
could decrease production of nasal secretions
What are the side effects of treatment of asthma using muscarinic antagonist?
Atropine side effects - Blocking parts of parasympathetic system
Blurred vision
Dry Mouth
Tachycardia
Constipation
Urinary retention
What are the side effects of using Beta(2) agonists?
Increased blood flow to skeletal muscle (decreased peripheral resistance) = tremor
Tachycardia (depending on dose) = increased heart rate
What are the adverse effects of Nasal Decongestants?
Decreased blood flow to skin and gut -> increased peripheral resistance -> increased blood pressure
Also “rebound congestion” – Rhinitis medicamentosa due to receptor down-regulation
What are nasal decongestants?
Nasal decongestants are
alpha-adrenoceptor agonists,
e.g. phenylephrine (pseudoephedrine)
Increasing airway patency and reducing nasal congestion.
How can we limit adverse effects of nasal decongestants?
local administration:
give it as a nasal spray
But beware of adverse effect of rebound congestion (Rhinitis medicamentosa) when used for too long
What must we target to treat as asthma?
The lungs To cause bronchodilation in the lungs = sympathetic system = Open airways even more
= Reduce bronchoconstriction
How can we treat asthma in terms of moderating the systems?
decreasing activity of the parasympathetic nervous system
OR
by increasing activity of the sympathetic nervous system
What can we give to decrease the activity of the parasympathetic nervous system?
Give a muscarinic antagonist,
e.g. ipratropium bromide or tiotropium bromide
We can Treat asthma by increasing activity of the sympathetic nervous system but what can we give to boost this?
Beta(2) agonists cause bronchodilation
e.g. salbutamol
How can we limit the adverse effects of Beta(2) agonists?
by local administration:
aerosol inhaler
e.g. salbutamol
What can we give if an aerosol inhaler does not work to treat asthma alone?
the muscarinic antagonist ipratropium bromide (muscarinic antagonist) can also be used as an aerosol to provide local effects but salbutamol is the preferred treatment
What do Nasal decongestants are
alpha-adrenoceptor agonists, e.g. phenylephrine (pseudoephedrine) target?
The radical muscles, Iris, Salivary glands, blood vessels, head and neck of the sympathetic system to contract secretion
What system does nasal decongestants cause adverse effects on?
Sympathetic
What pathway does alpha-1 adrenoceptors take And what does it cause?
Ca2+ pathway:
Phospholipase C coverts PIP2 to IP3 and DAG which causes the release of Ca2+ which causes smooth muscle contraction
Describe the pathways of alpha-2 adrenoceptors (2)
Inhibits the release of Ca2+
Inhibits cAMP production which causes smooth muscle contraction
Describe the pathway of Beta adrenoceptors
Takes cAMP pathway and causes heart muscle contraction, smooth muscle relaxation and glycogenolysis
What are the hallmarks of cancer?
- Self-sufficiency in growth signals
- Insensitivity to growth inhibition signals
- Evasion of apoptosis
- Limitless replication potential
- Neo-angiogenesis
- Tissue invasion and metastasis
With regard to self-sufficiency in growth signals what causes the hallmarks of cancer?
1) Increased exposure to growth factors
2) increased growth factor receptor expression on the tumour cell
3) genetic change(s) in a growth factor response pathway resulting in “always on” signalling.
What causes Insensitivity to growth inhibition signals?
The loss of function of a tumour suppressor gene e.g. P53 or RB
What causes the evasion of apoptosis?
Due to the gain of function the genes involved in the negative regulation of apoptosis
BCL2 or a loss of function of genes involved in the positive regulation of apoptosis e.g., BAX or P53
What causes limitless replication potential?
he activation of telomerase, the enzyme responsible for the maintenance of telomeres (the structures found at the ends of all linear chromosomes).
Activation of this enzyme prevents cells from reaching their notional “Hayflick limit” i.e., the point at which a cell can no longer divide.
What causes Neo-angiogenesis?
the process of new blood vessel production within and around a tumour. This is necessary to provide the tumour with nutrients and oxygen.
The process of neoangiogenesis is multi-factorial but it typically requires the tumour to produce vascular endothelial growth factor (VEGF).
What causes tissue invasion and metastasis?
The ability of tumour cells to invade tissues and move to distant sites (metastasis) is promoted by many different cellular events but neo-angiogenesis certainly aids the process of invasion and metastasis.
What are the enabling characteristics of cancer?
- Genome instability and mutation
- Tumour-promoting inflammation
What are regarded as the emerging hallmarks of cancer?
- Altered cellular energetics e.g., a switch to glycolysis for energy production
- Avoidance of immune cell recognition and destruction
What is an oncogene?
An oncogene is characterised by a gain of function mutation in a gene usually involved in the positive regulation of growth.
These mutations are dominant i.e., only need to occur in one allele.
What is a tumour suppressor gene?
A tumour suppressor gene is characterised by a loss of function mutation in a gene usually involved in the negative regulation of growth.
These mutations are recessive i.e., need to occur in both alleles in order to lose the tumour suppressive function.
What is a proto-oncogene?
This is a wild-type (unmutated) form of a gene that positively regulates growth (or inhibits cell death).
It has the potential to become an oncogene if it becomes mutated (or is aberrantly over-expressed).
What are mutator genes?
These are genes that are normally responsible for DNA repair. If they become mutated or deleted this leads to an increase in the frequency of genetic errors (mutations) that accumulate in the cancer cell.
What causes chronic myeloid leukaemia (CML)?
a translocation event between chromosome 9 and 22 (t9 and t20)
What does chronic myeloid leukaemia result in?
The production of a unique tyrosine kinase termed BCR-ABL
What is Imatinib?
selective inhibitor of this kinase and was the first example of the successful targeting of a genetic lesion in a human cancer
What do 15-30% of patients with invasive breast cancer over express?
The human epidermal growth factor 2 (HER2)
What targets the over-expression of HER2?
1) blocking the downstream signalling of HER2 (using Trastuzumab, also known as Herceptin)
2) blocking the dimerisation of HER2 (using Pertuzumab)
As well as targetting HER2, what can trastuzumab also do?
used as a targeting agent for chemotherapy in the form of an antibody drug conjugate (ADC) – Trastuzumab linked to the microtubule inhibitor, DM-1.
What do most tumours over express?
the anti-apoptotic protein BCL2
What targets BCL2 when it is over-expressed by tumours?
BH3 mimetic drug - Venetoclax
Give an example of when Venetoclax has led to mpressive responses and long-term remissions.
chronic lymphocytic leukaemia (CLL)
