Biochemistry + Cell Biology Flashcards
What are isotopes?
Molecules with the same chemical formulae but different structural formulae
- different numbers of neutrons
- are naturally occurring + many are radioactive
What does it mean that isotopes are radioactive?
They decay spontaneously but remain stable
What can radioactive isotopes be used to determine?
(14C) can be used to determine the age of organic artefacts = radiocarbon dating
- measures decay of 14C by its fixed half life
What is the simplest form of carbohydrates?
Saccharides
1 saccharide present = monosaccharide
What are monosaccharides used for and what is their chemical formulae? List some examples
Used for cellular energy and building nucleic acids
- (CH2O)n where n = 3/5/6
- glucose, fructose, galactose
Name of carbohydrate when there are…
1. 2 saccharides present
2. 3-10 saccharides present
3. >10 saccharides present
- Disaccharide
- Oligosaccharide
- Polysaccharide (starch)
List the key disaccharides and what they are formed of
Sucrose = glucose + fructose
Lactose = glucose + galactose
Maltose = 2x glucose
What causes a lactose intolerance?
Lactose is not broken down by lactase + utilised by gut flora
What are the main uses of lipids?
- long term energy storage
- protection/ insulation
- neuron myelination
- absorption of fat-soluble vitamins (A,D,E,K)
- hormone production (oestrogen, testosterone)
- cell membranes (lipid bilayers)
What is the simplest forms of lipids?
Fatty acids compounds which are organic chains with a functional carboxyl group
E.g. butyric acid (butanoic acid) = 4 carbon atoms or palmitic acid (hexadecenoic acid) = 16 carbon atoms
What are the 3 sub-groups of lipids?
Triglyceride
Phospholipids
Sterols
What are triglycerides?
Glycerol backbone with 3 bonded fatty acids (carboxyl to hydroxyl group bond)
- can be saturated (all single carbon bonds) or unsaturated (at least 1 carbon double bond)
What are the 2 geometries that doubles bonds can be in triglycerides?
Cis = same side of double bond
Trans = opposite sides of double bonds
- trans fats = linked to increased cholesterol levels + onset of CV diseases
What are phospholipids?
Glycerol group with associated phosphate group and only 2 fatty acids
- arranged into bilayers = help transport of fats in blood
- have a hydrophilic head + hydrophobic tail
What are sterols?
Cyclic organic compound found in grains, nuts, seeds etc
- sub group of steroids e.g. cholesterol
What are proteinogenic amino acids?
Amino acids found in nature - only 20 genetically encoded in DNA
- vital in formation of enzymes, antibodies, hormones, structural proteins + receptors
What is the structure of proteins?
Made up of a long chain of alpha amino acids joined by peptide bonds (written N-terminus to C-terminus)
- all have same configurations - L-alpha-amino acids (apart from glycine so no chirality)
- peptide bond between carboxyl + amine groups
List and describe the 4 levels of protein structure
Primary: amino acid sequence from N- to C- terminus
Secondary: alpha helix + beta sheets via hydrogen bonding
Tertiary: overall protein conformation caused by side chain interactions (folding of polypeptide chain into 3D shape)
Quaternary: higher order structure e.g. dimers (multiple subunits joined)
What is an alpha helix?
Each C=O (residue i) forms a hydrogen bond with the amide hydrogen of residue i + 4
- formed between the carboxyl + amino groups
What is a beta sheet?
The angular conformation of a peptide chain causes a ‘zig-zag’ shape
- backbone is able to form hydrogen bonds between each segment
- exist in either a parallel or anti-parallel structure
What is avogrados constant + what does molar conc refer to?
6.02 x 10^23 = 1 mole
- molar conc refers to the number of moles of a substance in a defined volume
- where 1 mole of a molecule is always its molecular weight in grams
What are the 3 main functional groups of compounds?
- Hydroxyl (OH)
- Amine (H2N)
- Carbonyl (COH)
What is an aromatic ring + pi pi stacking?
Every other carbon has a double bond
- very stable structures
- many amino acids have these
Pi Pi stacking is when aromatic rings slightly offset each other so don’t repel
List the equations linking mass, molecule weight (MW), moles
Mass = molecular weight x moles
Molecular weight = mass / moles
Moles = mass / molecular weight
Define the 1st Law of Thermodynamics
The total amount of energy within a system and its surroundings is constant
Energy can neither be created nor destroyed
Define the 2nd Law of Thermodynamics
The total entropy (level of disorder) of a system and its surroundings always increases
Define the 3rd Law of Thermodynamics
The entropy of a system approaches a constant value when its temperature approaches absolute zero
What do the terms system and surroundings refer to in the laws?
System = whatever part of the universe we are interested in
- closed = cannot exchange matter across boundaries
- open = can pass matter back and forth
Surroundings = everything that surrounds the system
What is enthalpy + give the equation
Enthalpy is the heat storage capacity of a system
Enthalpy = internal energy + (pressure x volume)
H (J or J/mol) = E (J) + (P (Pa) x V (m3))
What are exothermic and endothermic reactions?
Exothermic = release of heat energy during a reaction
Endothermic = heat energy is absorbed during a reaction
What does entropy refer to?
Refers to the level of disorder in a system (how energy is distributed)
Solid = low entropy
Liquid = entropy increasing
Gas = high entropy
What is Gibbs Free Energy (G)? Give the equation
The amount of energy available to do work
- tells us if a biochemical process will occur on its own (spontaneously or not)
Gibbs Free Energy (kJ/mol) = change in enthalpy (J) - (Temperature (K) x change in entropy (J/K))
What does it mean when…
G < 0
G > 0
When G < 0 = a spontaneous process
When G > 0 = not a spontaneous process
What’s the conversion for degrees C to Kelvin
37 degrees = 310 Kelvin
What are coupled reactions for?
Fundamental to metabolism
- reaction cannot occur spontaneously if G is > 0 so an energy input is required
What are the 2 types of coupled reactions + give the examples?
Anabolic reaction = constructing molecules from smaller units
- 2 x glycine = glyclglycine
- endergonic (unfavourable)
Catabolic reaction = breaking down molecules to form smaller subunits
- ATP + Water = ADP + Pi
- exergonic (favourable)
What do activated carriers do (thermodynamics)?
- Energy released by oxidation of organic molecules can be stored, preventing it being lost as heat
- Energy is stored in activated carriers, as either transferable chemical group or electrons
- Then used for endergonic reactions or other cell activities
What is ATP? - not the acronym but what does it drive etc
Most important activated carrier molecule
- the phosphorus-oxide bonds assists in storing lots of energy
- drives non-energetically favourable (endergonic) reactions in our body
What are the energy losses/ gain from ATP re-synthesis + hydrolysis?
ATP re-synthesis:
- G = +30.5 kJ/mol
ATP hydrolysis:
- G = -30.5 kJ/mol
How do cells obtain energy?
Via the oxidation of organic molecules (food)
- energy is stored in covalent bonds of these organic molecules
What do the terms oxidation + reduction mean?
Oxidation = removal (loss) of electrons from a molecule
Reduction = addition (gain) of electrons from a molecule
- electrons are never lost, so if one molecule is oxidised, the other must be reduced
What are NADH + NADPH + what are they involved in?
NAD+ = nicotinamide adenine dinucleotide
NADP+ = nicotinamide adenine dinucleotide phosphate
- they pick up energy in the form of 2 high energy electrons and a proton (a hydride ion H-)
What type of proteins are enzymes?
Globular proteins - polypeptide chain folds up into a compact shape, like a ball, with an irregular surface
- their tertiary / quaternary structures are quite complex
What are the active and inactive forms of enzymes called?
Active = holoenzyme
Inactive = apoenzyme
Briefly explain enzymes involvement in diseases
A malfunction in enzyme activity disrupts homeostasis
- DNA mutations resulting in under or overproduction/ deletion in a single critical enzyme results in disease
- a single amino acid substitution = destabilise protein structure/ disrupt binding etc
What are the 2 key theories of enzyme binding?
- Lock + Key Model = geometric complementarity, considered rigid + fixed
- Induced Fit Model = induces a conformational change on binding (more supported)
What determines substrate specificity?
Amino acid residues
What are the 2 sites of binding?
Active site - non-polar environment that enhances binding
Allosteric site - induces a conformational change as mechanism of regulation
Define the following terms…
1. Cofactor
2. Coenzyme
3. A prosthetic group
Cofactors = any factor essentially required for enzyme activity or protein function (inorganic)
Coenzyme = cofactor which is directly involved in enzyme catalysed reaction (organic)
A prosthetic group = covalently associated non-protein constituent required for a particular function
What is the common enzyme inorganic cofactor?
Metal co-factors otherwise called metalloenzymes
- cannot function without a metal ion in active site
What is an example of co-enzyme deficiency?
Scurvy - connective tissue disease due to lack of vitamin C
What is the transition state in relation to enzymes reaction?
Transient molecular state that is no longer substrate, but not yet product
Name some factors that cause activation energy to be lowered
Binding
Spatial + chemical complementarity
Induced fit
Specific amino acid R-groups
Orientation
Microenvironment (hydrophobic so no water)
Describe the 4 main catalytic mechanisms
- Metal ion catalysis - metal ions in active site
- Covalent catalysis - covalent bond between substrate + enzyme
- Catalysis by approximation - easier as come closer together
- Acid-base catalysis - use side chains to catalyse (chymotrypsin)
What is the equation for enzyme kinetics?
Rate of reaction is called velocity (V0)
= amount of substrate (S) converted to product (P) per unit of time
Describe the rules about the relationship between S and V0 (enzyme reaction rates)
Doubling S also doubles V0
Enzyme saturation means no further increase in V0
Reaction rate dependant on P dissociation from enzyme
Double E will double V0
Describe the Michaelis- Menten graph (enzyme kinetics)
Shows substrate conc on X against reaction rate on Y
- Vmax = highest rate of reaction
- Km (michaelis constant) is half Vmax and is a measure of enzyme affinity for substrate
- a low Km indicates weak binding and vice versa
What is the effect of increasing temp + pH on enzyme activity?
Rise in temp = increased thermal energy = overcome activation energy = increase in rate
Beyond optimal temp = breaks multiple weak bonds = alters active site = denaturation
(pH)
Small deviations results in decrease activity (ionisation of groups in active site)
- large deviations - denaturation
What are the 3 types of reversible inhibitors for enzymes?
Competitive
Non-competitive
Uncompetitive
How do competitive inhibitors work?
- they have some affinity for active site
- similar shape/ binding properties as substrate
- substrate is competing for access to active sites
- therefore reduced rate of activity
- can be overcome by increasing substrate levels
- Km increases but Vmax stays same
How do non-competitive inhibitors work?
Binds to allosetric site
- conformation of active site changes so substrate cannot bind
Km stays the same but Vmax is reduced
How does a uncompetitive inhibitor work?
Binds only when enzymes-substrate complex is formed
- binds to allosteric site
Vmax is much lower + Km is lower
- so enzyme has better affinity for substrate (substrate is always bound to active site)
What are the 3 other types of regulation of enzyme activity?
- Reversible covalent modification
- Proteolytic activation
- Apoptosis (programmed cell death)
What is glycolysis, where does it occur?
Oxidation of sugars
- occurs in cytosol of cells
- ATP is used in reactions 1 + 3 to phosphorylate the sugar + couple the reaction to make it energetically favourable
- NAD is reduced
- steps 1-5 = energy investment phase
- steps 6-10 = energy generation phase
- end with 2 molecules of pyruvate
Key Step
Explain step 3 of glycolysis - (PFK)
PFK (phosphofructokinase) is the most important regulatory + rate limiting enzyme in glycolysis (allosteric enzyme)
- activated by AMP + F 2, 6 BP and is inhibited by ATP, low pH + citrate
- low activity of PFK = slower glycolysis + vice versa
- causes shift in graph to left = more active PFK
- ATP is broken down to form ADP + Pi in this step
Key Step
Explain step 6 of glycolysis
- enzyme 1 binds to G3-P + catalyses its oxidation
- NAD+ accepts the electrons and is reduced
- enzyme 1 becomes displaced by Pi to create a high energy sugar-phosphate (1,3-biphosphoglycerate)
Key Step
Explain step 7 of glycolysis
- the sugar-phosphate intermediate binds to enzyme 2 which catalyses the transfer of Pi to ADP, creating ATP + generates carboxylic acid (3-phosphoglycerate)
(Extra Step 11 of glycolysis)
What happens when no O2 is available as an electron acceptor
NAD+ must be regenerated for glycolysis to continue
- When no O2 is available to act as an electron acceptor, NADH passes electrons to pyruvate which forms lactate
What is the net ATP gain of glycolysis?
2 ATP
After glycolysis, where do the products of pyruvate go?
Moves into the mitochondria where it is decarboxylated to form acetyl CoA
How are fats (lipids) oxidised to form acetyl CoA?
Beta Oxidation
- Fatty acyls (FA’s) are converted to fatty acetyl CoA + transported to mitochondria
- then enters beta oxidation
- this is a series of 4 enzymatic reactions which reduced the number of carbons on fatty acyl CoA (2 per decarboxylation)
= produces acetyl CoA, 1 NADH + FADH2
- continues until fatty acid is completely degraded
Describe the TCA (KREBS) Cycle
- Acetyl CoA (2C) is transferred to a 4C oxaloacetate to form the 6C tricarboxyllic acid (citric acid)
- following series of 8 reactions (4 oxidation) regenerate oxaloacetate
- energy release is captured in… 3xNADH, 1xFADH2, 1xGTP
How can the TCA cycle be controlled?
2 enzymes can be allosterically regulated…
- isocitrate dehydrogenase = stimulated by ADP, NAD+ and inhibited by ATP + NADH
- alpha ketoglutarate = stimulated by ADP and inhibited by succinyl CoA, ATP + NADH
Where does amino acid metabolism occur + what parts of amino acid are used to generate energy?
At the liver
- amino acids can be degraded + used as fuels
- nitrogen (amine group) cannot be used to generate energy so must be removed = leaving carbon skeleton for use
Step 1 amino acid metabolism
Explain transamination
Amino group of amino acid is transferred to keto acid
- typically then transferred to alpha ketogluterate
- this is accepted by the first keto carbon
(catalysed by enzymes called aminotransferases)
- oxaloacetate (another keto acid) can also accept the amino group
- this forms aspartate = required for urea formation + can be transaminated to form glutamate
Step 2 of amino acid metabolism
Explain deamination
(Glutamate has amino group)
- amino group is removed = deamination
- redox reaction catalysed by glutamate dehydrogenase
- removal of amino group as ammonia (TOXIC)
After ammonia is formed in mitochondria, what happens to it?
UREA CYCLE
- apartate comes from transamination reactions
- fumerate can be used in TCA cycle or to generate glucose
- urea enters blood + is transported to kidneys
What happens to the carbon skeletons?
Start with 20 amino acids
- 7 become metabolic intermediates
- Leu + Lys become ketogenic amino acids
- Ile, Phe, Try + Tyr = Keto + glucogenic amino acids
- glucogenic amino acids = 14 other amino acids
Some amino acid metabolism occurs in the muscle…
Glucose-Alanine Cycle
Muscle used BCAAs for fuel during prolonged exercise
- muscle doesn’t have urea cycle (no enzymes for it)
So buildup of glutamate causes alanine to be formed
- alanine is released into blood + taken up by liver
- alanine is transaminated + amino group is removed
What is gluconeogenesis?
Formation of glucose from non-carbohydrate sources
What are the main non-carb precursors for gluconeogenesis?
Where does it occur?
Lactate, glucogenic amino acids + glycerol
Occurs in liver = responsible for maintaining blood glucose
- essentially reversal of glycolysis = energetically favourable
Why do we have mitochondria?
Endosymbiosis
- prokaryote grows
- plasma membrane forms infoldings + pinches off in places forming membrane around organelles/ endomembrane system
- engulfs aerobic bacterium
What makes mitochondria special - what do they have?
- own genome
- own symbiotic machinery = making RNA + proteins
Describe the mitochondrial genome
Single circular chromosome in mitochondrial matrix
- 37 genes in total (2 rRNA, 22 tRNA, 13 protein encoding)
- mitochondrial DNA = oocytes
What’s do the terms fusion and fission mean?
Fusion = join
Fission = break apart
How are proteins transported into mitochondria?
Must have signal sequence to go into mitochondria
- enter via translocases of outer + inner membrane = TOM/TIM
- Signal sequence binds to import receptors
- Insertion into membrane by TOM complex
- Translocation into matrix by TIM23 complex
- Cleave by signal peptidase
How does pyruvate move into mitochondria?
Mitochondrial membrane contains porins (small channel proteins)
- pyruvate passes into matrix via mitochondrial pyruvate carriers
How do fatty acids move into mitochondria?
Beta oxidation cycles
- acyl-CoA combines with carnitine in cytosol to form acyl-carnitine
- this can move into inter membrane space from cytosol via CPT1
- then moves into matrix via a translocase + breaks apart releasing acyl-CoA
How does NADH move into the mitochondria?
Malate aspartate shuffle
1. NADH is oxidised to NAD via malate dehydrogenase
2. Oxaloacetate is reduced to form malate
3. Malate can pass from cytosol to inner mitochondrial matrix via porins
4. Inside mitochondria, malate is dehydrogenated to form oxaloacetate which reduces NAD to NADH
Give a brief overview of the electron transport chain - what enters, what is formed etc?
Involves 4 protein complexes
- high energy electrons, carried by NADH + FADH2, are passed into ETC + accepted by series of carriers
- high energy electrons lose energy as are passed along chain
- energy is used to pump protons (H+) across inner membrane of mitochondria
- oxygen accepts electrons, forming water
Explain what occurs at complex 1 of ETC
Called NADH-CoQ reductase (largest complex)
- NADH transfers its electrons into complex 1
- complex 1 passes electrons along with 2H+ to CoQ to form CoQH2
- energy released is used to pump 4H+ across membrane into inter membrane space
Explain what occurs at complex 2 of ETC
Called succinate-CoQ reductase
- (step 6 of TCA cycle) - succinate is oxidised to form fumerate + FADH2
- FADH2 remains coupled to succinate dehydrogenase + then immediately passed into complex 2 to Fe-S clusters
- electrons are ultimately passed to CoQ along with 2H+ to form CoQH2
- FAD is recycled
- H+ are not pumped across membrane as not enough energy is released
Explain what occurs at complex 3 of ETC
Called cytochrome c reductase
- CoQH2 from either complex 1/2 donate their electrons to complex 3 via Fe-S clusters and then to cytochromes
- the 2 protons are released into intermembrane space
- each electrons is passed to a cytochrome c = 2 reduced cytochrome c molecules being formed
- energy released pumps 2 more H+ across membrane
Explain what occurs at complex 4 of ETC
Called cytochrome c oxidase
- 4 electrons enter at complex 4 which couple with 4 protons + some oxygen to form 2 molecules of water
- 2 x electrons per NADH (2 cytochrome c carriers) = accepted by 1/2 oxygen + 1 proton = water ^^^
Describe the steps of oxidative phosphorylation + ATP synthase role
- Protons enter ATP synthase through F0 region
- Protons move to c subunits of F0 causing them to rotate (mechanical energy) then exit into matrix
- In turn, the y subunit (shaft) rotates very quickly
- ADP + Pi bind to specific sites between alpha + beta subunits
- When the y subunit rotates it causes a conformational change to F1 region + catalyses formation of ATP from ADP + Pi
List some key functions of plasma membranes
- physical + chemical barrier
- communication
- recognition
- energy conversion
- platform for cellular processes
Describe the structure of plasma membrane
Very thin film of lipids + proteins held via non-covalent interactions
- impermeable barrier to water soluble (polar) molecules
- phospholipids = amphipathic
- two HYDROPHOBIC fatty acid chains + a phosphate containing HYDROPHILIC head
What are fatty acids?
Long hydrocarbon chains with a carboxyl end (COOH) and a methyl end (CH3)
- can be free or covalently bonded to glycerol via ester linkage
What are some other types of membrane lipids?
Sterols (cholesterol) = rigid rings attached to hydroxyl group + short fatty acid
Sphingolipids = sphingosine backbone (first carbon attached to fatty acid + second attached to amine group)
What is important about polar/ non-polar molecules regarding thermodynamics in lipid bilayers?
Polar molecules can dissolve = energetically favourable, more disordered = increased entropy
Non-polar molecules cannot dissolve = energetically unfavourable, more ordered = decreased entropy
How do lipid bilayers form?
The amphipathic nature
- to avoid water forming cage structures around hydrophobic regions, the hydrophobic tails cluster together, exposing only the hydrophilic head to water
What can regulate the fluidity of lipid bilayers?
- Cis double bond make phospholipids more difficult to pack together + makes it thinner
- cholesterol
What are lipid rafts in membranes?
Locations in membranes that are more rigid than rest of membrane
- Van der Waals interactions are not strong enough to hold molecules together
- sphingolipids have long + saturated fatty acid chains and the attractive forces are strong enough to hold adjacent molecules together = rafts
- proteins often congregate here in prep for vesicular budding + transport
What are glycoproteins, glycolipids + glycocalyx?
Glycoproteins = proteins glycosylated in RER + golgi
Glycolipids = lipids from SER glycosylated in Golgi
- always found on the non-cytoplasmic side of membrane
= forms a CHO coating to the membrane termed the glycocalyx = protective
What are the 2 types of membrane proteins?
Transmembrane proteins
- spans membrane only once (single pass)
- e.g. alpha helix / beta sheet
Peripheral membrane proteins
- span membrane multiple times (pores/channels) = multi pass
- embedded in membrane
- or covalently bonded to lipid in membrane
What are the 4 groups of molecules and their transport across membranes?
Hydrophobic molecules = non-polar so can dissolve in bilayer + diffuse across
Small, uncharged polar molecules = OK to move across, just very slowly
Large, uncharged, polar molecules = need a transporter of some sort
Ions = need specialist mechanism
Explain the difference between carrier and channel proteins
Carrier = bind a solute + undergo a conformational change to transfer across membrane
Channel = interact only weakly with a solute - form an aqueous pore that solutes can pass through quickly
What are the 2 mechanisms by which eukaryotes carry out active transport?
- Coupled carriers (secondary active transport)
Symporters
- uses stored energy from electrochemical Na+ gradient + free energy released from transport of Na+
- energy is used to pump glucose in same direction (against conc gradient)
Antiporters
- transport solutes in different directions e.g. aspartate cycle - ATP driven pumps (primary active transport)
- Na+/K+ pump
- uses free energy released by ATP hydrolysis to pump 3Na+ out of cell + 2K+ in to cell
How is the acidity in a lysosome maintained?
Through pumping H+ ions across the membrane via an ATPase pump
- example of active transport
What are the 3 kinds of gated ion channels?
Voltage gated = open/close via stimulation from neurons
Ligand-gated = open/close via binding of ligand
Mechanically gated = open/close based on physical disruption
What is a cytoskeleton?
A large dynamic network of protein filaments that supports the cell
What are the 3 main functions of the cytoskeleton?
Shape - cell shape, position of organelles, organisation of cytoplasm
Strength - prevents cell from being damaged
Movement - provides a mechanism by which things can move within a cell + by which cells themselves can move
What are the 3 main types of protein filament in cytoskeleton + their roles?
Actin filaments = shape of cells surface, whole-cell locomotion, pinching one cell into two
Microtubules = position of organelles, intracellular transport, mitotic spindles
Intermediate filaments = mechanical strength
Explain the role of cytoskeletal filaments in the small intestine
- actin filaments enable microvilli formation (important for absorption)
- also creates a physical barrier by connecting to cell junctions
- Intermediate filaments anchor across the cell providing structure + strength via cell junctions called desmosomes
- microtubules run from basal to apical surfaces providing a intracellular transport network (polarity)
How are actin filaments formed?
By polymerisation of globular actin monomers (G-actin)
- non covalent bonds between adjacent molecules (dimer, then a trimer)
- the monomers form a protofilament
- 2 protofilaments twist to form a helix (F-actin)
- F-actin has a large cleft where ATP binds
What is nucleation?
The first step in the formation of a new structure via self-assembly
- 2 ATP G-actin subunits bind to each other weakly
- when 3 ATP G actin subunits form a trimer it acts as a nucleus from which a larger filament can form
- polymerisation + depolymerisation can occur at both ends but assembly is preferred at + end and disassembly at - end
What does treadmilling refer to?
Subunits are added much faster at + end and dissociate much faster at - end due to differences in Cc
- causes + end to grow and - end to shrink
- the overall length of the F-actin therefore remains constant = essential for cell motility + alterations in cell shape
What are the 2 types of protrusions that are important in actins role in cell motility?
- Lamellipodia = actin filaments in branched network
- Filopodia = narrow, finger like protrusions where actin filaments are in bundles
How are microtubules formed?
By the polymerisation of tubulin heterodimers consisting of alpha tubulin and beta tubulin
- non-covalent bonds between alpha + beta tubulin
- tubulin subunits form protofilaments
- 12 protofilaments form long hollow cylinders with a central lumen = essential for intracellular trafficking
What gives microtubules polarity (cell shape)
- beta tubulin exposed at + end
- alpha tubulin exposed at - end
Explain what is meant by dynamic instability regarding microtubules
Driven by the rate of tubulin addition + rate of GTP hydrolysis in beta tubulin
- if rate of GTP hydrolysis is greater than rate of tubulin addition = formation of a curved shape
- tubulin GDP dissociation is favourable + tend to extend/ shrink from + end
- the - end is stabilised at the microtubule organising centre (MTOC)
What is the MTOC actually called for microtubules?
The centrosome
- the - end remains anchored to the centrosome
- consists of 2 features = a pair of centrioles + pericentriolar material (matrix of proteins)
Describe what pericentriolar material is
The most important component in the synthesis of microtubules
- contains the amorphous material (a disorganised area of proteins)
- as well as containing the gamma-tubulin ring complex (y-TURC)
- y-TURC = multiple copies of y-tubulin complexed with numerous other proteins and is the specific site of nucleation for microtubules
What are the 2 associated motor proteins responsible for movement along microtubules?
- Kinesin = moves towards the + end of microtubules = Anterograde (away from centre)
- Dynein = moves towards the - end of microtubule = Retrograde (towards centre)
Describe the 6 step process of intracellular trafficking (microtubules)
- Starts with microtubule surface + kinesin molecules
- Lagging head is ATP bound + strongly associated with MT and the leading head is ADP bound + weakly associated with MT
- The hydrolysis of ATP causes lagging head to dissociate from MT
- Leading head binds to ATP and causes a conformational change
- Lagging head moves forward (hand-over-hand mechanism)
- Cargo gets transported along MT’s
Describe intermediate filaments
- Provide mechanical strength
- Multiple types composed of different protein subunits
- non polar + no associated motor proteins
- great heterogeneity
- hierarchail structure
- high tensile strength due to lateral association between tetramers
What are the 4 main categories of tissues?
- epithelial
- muscle
- nervous
- connective
Describe epithelial and connective tissues
Epithelial = sheets of cells that line surfaces of the body
- ECM (extra cellular matrix) is present as a very thin sheet called basal lamina
Connective = cells produce ECM and are sparsely populated within a dense matrix
What are the 3 types of cell junctions + what do they do?
Anchoring junctions = anchor cytoskeleton between cells/ between cells + ECM
Occluding junctions = prevents passage of ions + small molecules between cells
Communicating junctions = direct connections between cytoplasm of 2 cells
- allows passage of small water-soluble molecules from cell to cell
What are the 2 superfamilies to which cytoskeletal linked transmembrane proteins belong to? (Anchoring junctions)
Cadherins = cell-cell attachment
Integrins = cell-matrix attachment
Describe cadherins binding
C terminus of cadherins binds to cytoskeleton (inside cell)
N terminus binds to another cadherins = homophilic adhesion
- binding is dependant on extracellular Ca2+
Type of Cadherins binding 1
What are adherens junctions?
Connects actin filament bundles in 1 cell with that in next cell by cadherins
- intracellular binding to cytoskeleton is indirect as it uses adaptor proteins (catenin)
- these adaptor proteins assemble on C-terminus of cadherin + mediates binding
- catenines link cadherin to F actin
Type of Cadherins binding 2
What are desmosomes?
Connects intermediate filaments in 1 cell to those in next cell by cadherins
- also uses adapter proteins
- only found in vertebrates
- most plentiful in tissues subjected to great mechanical stress (heart + skin)
Describe integrins
They are composed of 2 glycoprotein subunits
- both subunits span the plasma membrane (single pass)
- small intracellular C terminus + large extracellular N terminus
Types of integrins binding 1
What are focal adhesions (also called actin-linked junctions)?
In the extracellular domain, they bind to a specific amino acid (AA) sequences in ECM proteins
- integrins binds to fibronectin (in ECM) which has a RGD sequence (arginine, glycine, aspartate)
- intracellular domain binds to a complex of several proteins which link to F actin (involve talin + Vinculin)
Typos of integrins binding 2
What are hemidesmosomes?
They connect the ECM to intermediate filaments within a cell
- use integrins in same way as focal adhesions
- most prominent cell-matrix junction in epithelial cells
- rely on specific integrins termed alpha6 beta 4 integrin
What are occluding junctions formed from + describe in greater detail what they are?
Also called tight junctions in which they seal gaps between epithelial cells
- ensures that molecules/ fluid entering cells at apical side cannot diffuse back peripherally
They are formed of sealing strands = long row of transmembrane homophilic adhesion proteins
What are 2 types of sealing strands in occluding junctions?
Claudins = essential mediators of tight junction formation - if knocked out of cells genetically then tight junctions will not form
Occludins = non-essential but important for limiting permeability of junction
Describe communicating junctions in greater detail
Provide channels which connect the cytoplasm of 2 cells
- permeable to small molecules up to 1000 Da (ions, AAs, sugars) so macromolecules cannot pass
- Normally gated = sensitive to voltage, pH, Ca2+, neurotransmitters etc)
What are the 2 proteins that span the gap in communicating junctions
The 2 membranes come very close together and the gap is spanned by channel forming proteins…
- Connexins = vertebrates
- Innexins = invertebrates
What is the extracellular matrix, what’s its role + what is it secreted by?
Intricate network of proteins, carbs + water (except bone as has no water)
- provides support for cells + tissue
- Is an important regulator for cellular signalling
It is secreted by cells (e.g. fibroblasts) in which its organisation is tissue dependant
What is the composition of the ECM - 3 major families of macromolecules/proteins?
- Glycosaminoglycans (GAGs) = oligosaccharides covalently bonded to a protein = proteoglycans
- Fibrous proteins (collagens)
- Glycoproteins
What do the terms histone and nucleosome mean?
Histone = protein that binds to DNA to form eukaryotic chromosomes
Nucleosome = a protein-DNA complex containing 8 histone proteins
- most basic level of chromosome packaging
Define the terms chromatin, chromosome and sister chromatids
Chromatin = repeating nucleosome structure like beads on a necklace
- also bound to non-histone proteins
Chromosomes = defined region of chromatin that carries info relating to a specific set of genes
- 23 pairs in nucleus
Sister chromatids = identical copies of same chromosome
- formed via DNA replication
- only seen during metaphase of mitosis
Describe the 4 main features of the nucleus
- Nuclear envelope that separates nucleus from cytoplasm
- Nuclear lamina that lines inside of envelope
- Inner + outer membrane forms phospholipid bilayer
- Perinuclear space between the 2 membranes
What is the nuclear pore complex (NPC)
It is an extremely large protein structure (quaternary) forming an aqueous channel that connects the nucleoplasm to the cytosol
- has a very ordered 8 fold symmetry
- as well as cytosolic fibrils (long proteins) that reach into the cytosol
- has a cage like structure = nuclear basket
Explain these 3 types of transport through a nuclear pore complex…
1. Molecules of size < 5000 Da
2. Proteins up to 60 KDa
3. Proteins > 60 KDA
- Freely diffuse through NPC
- Can diffuse through, just takes a lot longer
- Cannot diffuse through so requires a specialist mechanism + energy
What is a nuclear localisation signal?
They tell a protein to go into the nucleus
- proteins are synthesised on a cytosolic ribosome containing a NLS
What do nuclear import receptors do?
They recognise nuclear localisation signals
- nuclear import receptor binds to FG repeats on nucleoporins in the cytosolic domain of NPC
- proteins move through the NPC binding sequentially to FG repeats
Describe the process of nuclear import [12]
- Protein destined for nucleus is synthesised on an 80S ribosome with a nuclear localisation signal in its primary sequence
- Nuclear localisation signal is recognised by a nuclear import receptor and the import receptor binds to the localisation sequence and the protein itself
- The protein complex binds to cytosolic fibrils of nuclear pore complex
- The protein complex moves through the nuclear pore complex by binding, dissociating + rebinding with special nuclearporins containing FG repeats
- Protein complex arrives in nucleoplasm
- Ran-GTP binds to nuclear import receptor
- Nuclear import receptor releases its protein cargo into nucleoplasm
- Empty import receptor bound to Ran-GTP moves through nuclear pore complex to cytoplasm.
- Ran-GTP is bound by a ran binding protein, and it releases the nuclear import receptor
- Ran-GAP hydrolyses Ran-GTP to leave Ran-GDP + Pi
- Ran-GDP is transported from cytoplasm to nucleus through nuclear pore complex
- Ran-GEF coverts Ran-GDP to Ran-GTP
Describe the step of Nuclear Export [8]
- Ran-GTP binds to nuclear export receptor
- A protein to be exported from the nucleus binds to the NER
- Export receptor and its cargo bind to nucleoporins on nuclear basket
- Protein complex moves through nuclear pore complex by binding, dissociating + rebinding with special nucleoporins containing FG repeats
- Protein arrives in cytoplasm
- Ran-GTP is bound by a ran binding protein and Ran-GAP + GTP is hydrolysed to GDP
- NER releases its cargo + Ran-GDP
- Ran-GDP moves back through to the nucleoplasm through nuclear pore complex
What are the 2 types of chromatin in the nucleolus?
Heterochromatin = transcriptionally inactive + densely packed (dark regions) - hinders transcriptional machinery
Euchromatin = transcriptionally active + loosely packed (light regions)
What are the sizes of the large and small subunit of a ribosome + what are these made up of?
Large = 60S (3 pieces rRNA + 49 proteins)
Small = 40S (1 piece rRNA + 33 proteins)
Where are rRNA genes found, what ones are transcribed by what?
rRNA genes are clustered together on a DNA sequence as tandem repeats (adjacent to each other)
- speeds up rRNA transcription
- also spacer regions between tandem repeats = non-coding DNA sequence
18S, 5.8S and 28S rRNAs are transcribed in the nucleolus by RNA polymerase I
5S is transcribed in the nucleoplasm by RNA polymerase III then imported into nucleolus
In the nucleus, ribosomes are synthesised… describe ribosomal biogenesis [8]
- RNA polymerase I transcribes pre-rRNA inside nucleus
- Ribosomal proteins enter nucleus via nuclear pore complex + translocate to nucleolus
- Ribosomal protein associate with 45S pre-rRNA
- 90S pre-ribosome is formed
- SnoRNPS modify rRNA in 3 ways…
(i) Methylation of hydroxyl groups on ribose
(ii) conversion of uridine to pseudouridine
(iii) cleavage of 45S pre-rRNA by exo/endonucleases - RNA polymerase III transcribes 5S rRNA in nucleoplasm which is transported to nucleus
- 5S rRNA associates with 28S + 5.8S rRNA to form the 60S ribosomal subunit
- 40S and 60S subunits leave the nucleus using Ran-GTP mediated nuclear export
How do the following 3 methods of RNA modification work?
(i) Methylation of hydroxyl groups on ribose
(ii) conversion of uridine to pseudouridine
(iii) cleavage of 45S pre-rRNA by exo/endonucleases
(i) it stabilises RNA
(ii) isomerisation reaction improves the affinity of RNA to tRNA during protein synthesis
(iii) SnoRNPs contain exoribonucleases that cleave + removed externally transcribed spacers and endoribonucleaus that remove internally transcribed spacers
What are the key functions of the smooth endoplasmic reticulum?
- synthesis of lipids (phospholipids + cholesterol)
- synthesis of steroid hormones (testosterone) - adrenal cortex is rich in sER
- storage + release of Ca2+ from sarcoplasmic reticulum
- detoxification (alcohol) - lots in liver
What are the key functions of the rough endoplasmic reticulum?
Site of protein synthesis + processing for transmembrane proteins destined for…
- ER
- Golgi
- Plasma membrane
- Lysosomes
- Endosomes
What is co-translational translocation (protein synthesis)?
In mammalian cells most proteins are imported to ER during protein synthesis
- Co-translational = happening at same time as translation
- translocation = translocating to another part of the cell (ER)
One end of the protein is attached to the ribosome whilst the other inserts itself to the ER
When is post-translational translocation seen?
When a chaperone protein binds + escorts other proteins (preventing folding/damage)
- then imported to mitochondria via TIM/TOM complex
For co-translational translocation, the signal sequence must…
- Be recognised (by signal recognition particle/ SRP)
- Be recognised by the ER and embedded within the ER membrane (via SRP receptor + translocator)
- Be recognised (by signal recognition particle/ SRP)
Describe what a SNP is/ what do it does?
What it does
- It recognises the signal sequence on the N terminal of the growing polypeptide
It has 2 specific regions:
1. Translational pause domain = binds to ribosome + translation of domain is paused
2. Signal sequence binding pocket = binds to signal on protein itself
- Be recognised by the ER and embedded within the ER membrane (via SRP receptor + translocator)
Explain this process
- Ribosomes free in the cytosol start to translate the protein
- A signal sequence emerges from the ribosome which is recognised by SRP
- One part of the SRP binds to ribosome + one part binds to signal on protein
- This causes a pause in translation
- The ribosome (with help of SRP) moves to the membrane of ER
- SRP binds to a SRP receptor while the protein starts to be transported into a protein translocator
- The SRP + receptor are freed from the ribosome + separated = for use in new cycle
- Ribosome stays bound to ER membrane
- Translation will continue while protein is being translocated to ER
What happens after the ribosome has translated in co-translational translocation?
The mRNA is released back to the free ribosome pool
- the mRNA may still remain bound to the ER as it is translated by multiple ribosomes
- ultimately mRNA will be degraded
Once the protein has been translated + is in the ER, what are the 2 ways it will be modified?
- Fold into its 3D conformation via binding
- Glycosylation
What is protein glycosylation?
It refers to the addition of a sugar to a protein to form a glycoprotein
- a precursor oligosaccharide composed of 14 sugars is added to the N terminus of an asparagine side chain of the protein
It is very specific to proteins that enter the ER:
- quality control
- recognition
- protection
Why is glycosylation important, explain it?
Quality control - to recognise correctly folded (mature) proteins
- If the protein is correctly folded, 3x glucose are cleaved from N-linked oligosaccharide
- If it doesn’t fold correctly, glycosyl transferase enzyme adds a single glucose back
- Calnexin (chaperone protein) binds to unfolded protein to prevent aggregation
- Removal of the terminal glucose (enzyme called glucosidase) releases protein from calnexin
- Glycosyl transferase determines if protein is correctly folded, if not single glucose is added back
- If protein remains misfolded, then it will be released from ER + targeted for degradation within a cell
When misfolded proteins accumulate within a cell, what do it cause?
Causes ER stress and triggers an Unfolded Protein Response (UPR)
- Inhibit protein synthesis
- Degrades misfolded proteins
- Increase transcription of chaperones (BiP, Calreticulin, calnexin)
If problem persists it leads to apoptosis
What are the different ER receptors in unfolded protein response?
PERK = responsible for pausing translation
IRE1 + ATF6 = protein degradation + activation of genes to increase protein folding capacity
What are the 3 pathways in intracellular membrane traffic?
Secretory pathway = exocytosis
Endocytic pathway = endocytosis
Retrieval pathway
What is the movement of proteins in intracellular membrane traffic mediated by?
Vesicles from donor to target compartment by budding + fusion
- movement of vesicle is through movement on microtubules
What are the 3 types of vesicles in protein transport?
COPII coated vesicles - from the ER
COPI coated vesicles - from the Golgi
Clathrin coated vesicles - from plasma membrane (endocytosis) as well as between Golgi + endosomes
How do vesicles find the right membrane?
By the GTP binding protein called Rab-GTP
- found on the cytosolic surface of vesicles
- when it is GDP bound = inactive (in cytosol)
- when it is GTP bound = active (in vesicle membrane)
How is Rab-GTP formed?
GEF found on a vesicle membrane activates Rab-GDP in the cytosol to form Rab-GTP
- Rab-GTP then binds to a Rab effector protein on the target membrane = tethering of the vesicle to the target membrane
Describe the process of membrane fusion [7]
- At start the vesicle has the correct v-SNARE and Rab-GTP on surface
- It binds to the Rab effector protein on target membrane (tethering)
- Effector protein changes conformation and brings vesicle close to target membrane (docking)
- T-SNAREs and V-SNAREs intertwine = trans-SNARE complex
- Membranes can slip + fuse with each other = fusion of vesicle with target membrane
- (during process of docking + fusion) - Rab-GAP causes Rab to hydrolyse GTP to GDP
- Rab-GDP is then released from vesicle + bound by Rab-GTP dissociation inhibitor (GDI) to keep Rab inactive
Protein Sorting
What is the purpose of the further processing of oligosaccharide chains (by Golgi)?
- promote correct folding of proteins
- prevent unwanted aggregation
- act as signals for sorting and targeting to correct pathway
What are the 2 pathways within exocytosis for protein sorting?
It’s for if cargo is coming from ER through golgi to outside of cell
Constitutive secretory pathway
- vesicle reaches plasma membrane + is excreted from cell
Regulated secretory pathway
- vesicle is made but stored in cytosol
- only fuses with plasma membrane + release cargo upon receiving a signal (e.g. insulin)
- insulin is stored in vesicles + when blood sugar levels are high, vesicles move forward + release insulin into blood
What are lysosomes?
They are degradative organelles - digest unwanted material
- contain lots of enzymes to break down macromolecules
When are lysosomes active + how is their pH maintained?
Only active at pH 4.5-5 = important protective mechanism
- pH is maintained via vacuolar ATPase
What kind of vesicles must be used to transport proteins from Golgi to lysosomes?
Clathrin coated vesicles
- these are also mannose-6-phosphate tagged
What are endosomes, late endosomes and what forms endolysosomes?
Endosomes = term for saying cargo is packed in vesicles + proteins are destined to be degraded
Late endosomes = contain material that has been ingested by the cell
- fusion of late endosomes with lysosomes generates endolysosomes
Describe the endocytic pathway of protein sorting [7]
- Protein is made in ER and transported to golgi
- To be directed to lysosomes – they have to have M6P
- Within golgi there is addition of P-GlcNAc (phosphorylated N-acetylglucosamine) to protein
- Phosphate group is then transferred to mannose = M6P
- Protein is then packaged in a vesicle (clathrin coat)
- Buds off golgi + transported to fuse with early endosome
- Due to specific pH inside, proteins are going to dissociate from receptor = hydrolase enzyme
Retrieval pathway
After an endocytic vesicle fuses with an early endosome, the ingested material can either be…
Degraded - it moves towards the centre of the cell and membrane composition alters. It no longer recycles + starts degradation
Recycled - vesicles containing material for recycling to the plasma membrane bud off and fuse with a recycling endosome
What are the reasons for controlling cell growth + division?
- Embryogenesis
- early growth
- wound healing
- menstruation
- muscle growth
What are the 2 halves and the subunits within cell cycle?
Interphase (majority of time)
- G0, Gap 1, S phase, Gap 2
M phase (roughly 1 hour)
- prophase, prometaphase, metaphase, anaphase, telophase
What roughly occurs in the following stages…
1. M phase
2. Interphase
- G1 Phase
- S Phase
- G2 Phase
- Mitosis (nuclear division), usually ending in cell division (cytokinesis) (4)
2a. G1 = cell is metabolically active + growing (1)
2b. S phase = DNA replication takes place (2)
2c. G2 = cell growth continues + proteins are synthesised (3)
How is the cell cycle regulated?
By a series of control points
- START is a major control point, controlling the progression from G1 to S
- once they pass START they are committed to the cycle
- in most animal cells, the restriction point in late G1 functions like START
- if the appropriate growth factors are not present in G1 = progression stops at restriction point + enters resting G0 stage
What is the role of DNA damage checkpoints and spindle assembly checkpoint?
- Ensure that damaged DNA is not replicated + passed on to daughter cells
- Stops mitosis at metaphase if chromosomes are not properly aligned on spindle
What is the role of MCM helicase during cell cycle?
Bind either side of origin recognition complex + serve to open up DNA so DNA replication machinery is allowed in to replicate DNA
What are Cdk1 and Cyclin B - cell cycle?
Cdk1 = a protein kinase cell cycle regulator - adds a phosphate group as it is a kinase enzyme
Cyclin B = regulatory subunit required for catalytic activity of Cdk1 protein kinase
These 2 bind to form a maturation promoting factor (MPF) which is regulated by phosphorylation/ dephosphorylation of Cdk1
- when Cdk1 is phosphorylated = inactive form
- when Cdk1 is dephosphorylated = active form
Explain what occurs during G2 of the cell cycle regarding Cdk1 and Cyclin B
- Cyclin B is synthesised + forms complexes with Cdk1
- Cdk1 is phosphorylated by Wee1 and inhibited, this leads to the accumulation of inactive Cdk1/cyclin B complexes
- CDK1 is dephosphorylated by phosphatases + becomes active = cells progress through G2
- Cyclin B is then degraded by ubiquitin-mediated proteolysis
- The destructions of cyclin B inactivates Cdk1, so the cell exits mitosis + undergoes cytokineses = return to interphase
List the complexes of cyclins:cyclin-dependent kinases during the following stages…
1. Early G1
2. Past restriction point (for cell to go into S phase)
3. S phase
4. G2
5. M Phase
- Cyclin D binds to Cdk4 and 6
- Cyclin E + Cdk2
- Cyclin A + Cdk2
- Cyclin A + Cdk1
- Cyclin B + Cdk1
How is the activity of Cdks regulated?
- Cdk/Cyclin association is controlled by Cyclin synthesis + degradation
- Activation of Cdk/Cyclin complexes requires phosphorylation of threonine at position 160 (catalysed by CAK)
- Inhibitory phosphorylation of tyrosine near Cdk amino terminus (catalysed by Wee1) means Cdks are then activated by dephosphorylation by Cdc25 protein phosphatases
- Binding of inhibitory proteins - Cdk inhibitors (CKIs)
- 2 families = Ink4 + Cip/Kip
What does Cyclin D do and how is it synthesised?
It provides one link between growth factor signalling + cell cycle progression
- growth factors stimulate cyclin D synthesis through the Ras/Raf/MEK/ERK pathway
- it is synthesised as long as growth factors are present
What is Rb - cell cycle?
A substrate protein of Cdk4,6/Cyclin D complexes + is frequently mutated in many human tumours
- it is the prototype of a tumour suppressor gene, who’s inactivation leads to tumour development
What is Rb’s role during G0/ early G1?
It binds to E2F transcription factors, which suppresses expression of genes involved in cell cycle progression
Rb is then phosphorylated by Cdk4,6/Cyclin D as cells pass through the restriction point + dissociates from E2F, allowing transcription to proceed
How do cells progress through the restriction point?
Mediated by activation of Cdk2/Cyclin E complexes
- in G0 + early G1, Cdk2/Cyclin E is inhibited by p27 (part of Cip/Kip family)
Cyclin E synthesis is stimulated by E2F after Rb is phosphorylated + transcription of p27 is inhibited by growth factor signalling
- the resulting activation of Cdk2/Cyclin E leads to activation of MCM helicase + initiation of DNA replication
What mediates cell cycle arrest at DNA damage checkpoints?
Explain what happens
- Mediated by protein kinases (ATM ands ATR) that are activated in response to DNA damage
- They then activate a signalling pathway that leads to cell cycle arrest, DNA repair + sometimes apoptosis
What do the protein kinases ATM and ATR recognise what do this lead to?
ATM recognises double-strand breaks
ATR recongises single-stranded on unreplicated DNA
- they phosphorylate + activate the checkpoint kinases Chk1 and Chk2
- these then phosphorylate + inhibit Cdc25 phosphatases, which are required to activate Cdk1 and Cdk2
- inhibition of Cdk2 results in cell cycle arrest in G1 and S
- inhibition of Cdk1 results in arrest in G2
What is the role of p53 in G1 arrest?
It is a tumour suppressor protein - transcription factor whose increased expression leads to activation of Cdk inhibitor p21
- p21 inhibits Cdk2/Cyclin E complexes, leading to cell cycle arrest in G1
- It is phosphorylated by both ATM and Chk2
