Term
| Differences between prokaryotic cells and eukaryotic cells? |
|
Definition
| Eukaryotic are normally bigger, have a nucleus and membrane bound organelles |
|
|
Term
| Which two things make up the majority of the cell by %cell weight, and what else is found in the cell? |
|
Definition
Water (70%) and Macromolecules (26%)
Also Inorganic salts, sugars, amino acids, nucleotides, fatty acids and other small molecules |
|
|
Term
| What are the most common cellular ions? |
|
Definition
|
|
Term
| What different types of proteins are there? |
|
Definition
| Structural, enzymatic, import.export, signalling, motor, defence, attack (poison) |
|
|
Term
| What is the standard amino acid structure? |
|
Definition
|
|
Term
| How many different forms does the amino acid R group have? |
|
Definition
| 19 amino acids, and 1 imino acid |
|
|
Term
|
Definition
| Proline, and it is incapable of forming hydrogen bonds |
|
|
Term
| Which amino acids are aliphatic, and what properties do they have? |
|
Definition
Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine.
They are uncharged and hydrophobic, except glycine. |
|
|
Term
| Which amino acids are aromatic, and what properties do they have? |
|
Definition
Tyrosine. phenylalanine, tryptophan.
Tyrosine can form hydrogen bonds, and all three can stack against other planar groups. |
|
|
Term
| Which amino acids are polar, and what properties do they have? |
|
Definition
Serine, threonine, asparagine, glutamine.
They are hydrophilic, uncharged, have hydroxyl and amide groups, and can form hydrogen bonds. |
|
|
Term
| Which amino acids are charged and acidic, and what properties do they have? |
|
Definition
Aspartate, Glutamate.
They are negatively charged at neutral pH, can form hydrogen bonds, and can form slat bridges with positvely charged residues. |
|
|
Term
| Which amino acids are charged and basic, and what properties do they have? |
|
Definition
Lysine, Arginine, Histidine.
They are positevly charged at neutral pH, can form hydrogen bonds, and can form slat bridges with negatively charged residues. |
|
|
Term
| Why is cysteine unique and what can it do? |
|
Definition
| It is the only one with a sulphydryl group and is highly reactive, common at active sites, can form disulfide bridges, and is common at disulfide bridges. |
|
|
Term
| What makes amino acids enantiomers?q |
|
Definition
| They have an L and a D conformation, only L amino acids are in eukaryotic proteins |
|
|
Term
| What are the various protein structures? |
|
Definition
Primary structure - amino acid sequence
Secondary structure - arrangement of amino acids close to each other into defined structures
Tertiary structure - folding of protein into domains and overall structure
Quaternary structure - assembly of individual polypeptides into a multi-subunit protein |
|
|
Term
| How are peptide bonds formed? |
|
Definition
| By endergonic condensation reactions |
|
|
Term
| What sort of R group arrangements are favoured? |
|
Definition
|
|
Term
| What are disulfide bonds normally formed between, and where are they common/ |
|
Definition
| Cysteine residues, extracellular proteins |
|
|
Term
| What do secondary structures result from, and how are they stabilized? |
|
Definition
| Non-covalent interactions, repeating H+ bonds |
|
|
Term
| What is the alpha helix structure? |
|
Definition
| A right handed helix (obviously), with 3.6 amino acids every turn, H bonds between every 4th residue. Proline will induce a kink. |
|
|
Term
| What can disruppt alpha helices? |
|
Definition
| Large numbers of charged or bulky residues |
|
|
Term
| What is the beta sheet structure? |
|
Definition
| At least two beta strands with H bonds between adjacent strands, can be parralel or antiparrallel, with side chains protruding above and below. |
|
|
Term
| What happens when secondary structure combine? |
|
Definition
| They produce super-secondary structures known as motifs, such as beta barrels. |
|
|
Term
| What stabilizes tertiary structure? |
|
Definition
| hydrophobic effect, van der waals forces, salt bridges and H bonds, with large residues folding into domains |
|
|
Term
| What comprises the lipid bilayer? |
|
Definition
Phosphoglycerides/glycerophospholipids comprise most of the lipid bilayer
They are formed of glycerol, normally bound to 1 or 2 fatty acids, and a phosphate group |
|
|
Term
| What are head groups found in phosphoclycerides normally made of? |
|
Definition
–H (phosphatidic acid, PA)
–Ethanolamine (phosphatidyethanolamine, PE)
–Choline (phosphatidylcholine, PC)
–Serine (phosphatidylserine, PS)
–Glycerol (phosphatidyglycerol, PG)
–Phosphatidylglycerol (cardiolipin)
–Inositol (phosphatidylinositol, PI)
–Inositol 4,5 bis phosphate (phosphatidylinositol 4,5 bisphosphate, PIP2)
–Polyphosphoinositides are signalling molecules |
|
|
Term
| Where are phospholipids normally saturated? |
|
Definition
|
|
Term
| What remodels fatty acid groups? |
|
Definition
| phospholipasesand acyl transferases |
|
|
Term
|
Definition
Contain sphinosine
Simplest is ceramide
Sphingomyelin is an important constituent of myelin
Many contain carbohydrates
Found in the outer leaflet of the plasma membrane, concentrated in lipid rafts and calveolae, together with cholersterol are involved in compartmentalisation of membrane proteins
Lipid rafts are dynamic and involved in signalling events |
|
|
Term
| Where are phospholipids synthesized? |
|
Definition
| on the cytoplasmic face of membranes |
|
|
Term
| How do choline phospholipids make it into the outer leaflet? |
|
Definition
| They are flipped in by flippase enzymes |
|
|
Term
| Where do inositol and athanolamine headed phospholipids end up? |
|
Definition
| in the inner leaflet. No idea what they do there. Telling me that would be helpful, and we couldnt possibly have ANYTHING like that. No sir. |
|
|
Term
| What are red blood cell membranes rich in? |
|
Definition
| sphingomyelin and phosphatidylcholine |
|
|
Term
|
Definition
| specialised areas of plasma membranes rich in cholestrerol, they group together funcitonally related proteins. This is important in cell signalling |
|
|
Term
| What must cell compartment membranes be/ |
|
Definition
| Impermeable to hydrophilic molecules |
|
|
Term
| What is the outer mitochondrial membrane full off? |
|
Definition
|
|
Term
| What is the intermembrane space similar to? |
|
Definition
| the cytosol with chaperone proteins |
|
|
Term
| What is the inner mitochondrial membrane like? |
|
Definition
| convulted and impermeable, with cardiolipin, transporters to regulate ion flow |
|
|
Term
| Whats the endoplasmic reticulum like? |
|
Definition
Convoluted membrane with lumen
Contiguous with nuclear membrane
Largest internal membrane
Rough ER binds ribosomes, synthesizes compartmentalised proteins
Smooth ER synthesizes lipids and starts glycosylation
Transport vesicles bud off to transport cargo to golgi
Can store calcium and release in signal transduction |
|
|
Term
|
Definition
|
|
Term
|
Definition
| Organise transport vesicles and protein modification |
|
|
Term
|
Definition
–H+ pump lowers pH to 4.8 (vs 7.2 in cytosol)
–Acid hydrolases degrade proteins, lipids, complex carbohydrates, nucleic acids
–Fuse with autophagic vacuoles containing sequestered cytoplasmic material |
|
|
Term
|
Definition
–Oxidise fatty acids (glyoxysomes in plants)
–Decompose hydrogen peroxide via catalase
–Involved in lipid biosynthesis (cholesterol, plasmalogens)
–Sometimes has a crystalline core |
|
|
Term
| What is the cytosol like? |
|
Definition
Consists of soluble proteins, small molecules, ions water
Site of glycolysis, pentose phosphate pathway and biosynthesis of fatty acids |
|
|
Term
| What comprises the cytoskeleton? |
|
Definition
| actin filaments, intermediate filaments, microtubules |
|
|
Term
| What do actin filaments do? |
|
Definition
| maintain cell shape by resisting tension, move muscle cells, divide cells, move organelles |
|
|
Term
| What do intermediate filaments do and what are they made of? |
|
Definition
| Maintian cell shape by resisting tension, anchor nucleus and some other organelles, likely keratin or lamin or vimentin |
|
|
Term
| What do microtubules do and what aree they made of? |
|
Definition
| Maintain cell shape by resisting compression, move cells, move chromosomes, assist formation of cell plate, move organelles, provide tracks for intracellular transport, alpha and beta tubulin dimers |
|
|
Term
| How does the cytoskeleton interact with the extracellular matrix? v |
|
Definition
| via the plasma membrane spanning proteins |
|
|
Term
| Differential centrigugation, what do you do to do it? |
|
Definition
–Centrifugation through iso-osmotic buffer e.g. 0.25 sucrose, 5mM Tris-Cl, pH 7.2
–Different sedimentation rates depending on properties of particle e.g. density, shape
–Sequential centrifugation steps collecting sediment (pellet) and taking supernatant to next step |
|
|
Term
| How is Isopynic (equal density) centrifugation performed? |
|
Definition
–Create a density gradient from centrifugation of density gradient forming solution/suspension (Percoll, Nycodenz)
–Apply sample and centrifuge
–Organelles/membrane accumulate (band) where gradient density = their own
–Collect fractions and analyse
–Use “marker enzymes” to characterise fractions |
|
|
Term
| When does yeast reproduce sexually? w |
|
Definition
| when mating factors are present |
|
|
Term
| What does the nucleus contain? |
|
Definition
| Contains DNA, RNA and proteins (histones, RNA polymerases, transcription factors) |
|
|
Term
| How many membranes does the nucleus have? |
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
A network of intermediate filaments known as lamins underlie the inner nuclear membrane, found in eukaryotes that aren't yeast
It has roles in nuclear stability and positioning, spacing of nuclear pores and organisation of chromatin |
|
|
Term
|
Definition
Encoded by three genes in humans LMNA, LMNB1 and LMNB2
LMNA encodes the A type lamins (lamin A, lamin AD10, lamin C and lamin C2; Splice variants from the same gene )
LMNB1 and LMNB2 encode B type lamins |
|
|
Term
|
Definition
| Lamins form dimers, the dimers form tetramers, the tetramers assemble end to end into protofilaments (four protofilaments then associate to form a protofibril) |
|
|
Term
| What causes Hutchinson-Gilford Progeria Syndrome? |
|
Definition
Caused by a C(1824) – T mutation in the LMNA gene, this is a silent mutation. but creates a new splice site
This results in the lamin A missing 50 amino acids from the tail domain – with severe consequences |
|
|
Term
| Hutchinson-Gilford Progeria Syndrome symptoms? |
|
Definition
| “accelerated ageing disorder” poor growth, loss of subcutaneous fat, atherosclerosis, bone deformities, hair loss, sclerodermatous skin, cardiovascular disease |
|
|
Term
| What post-translational modifications does lamin A undergo? |
|
Definition
First, the C terminus of the lamin precursor protein is farnesylated, on a cysteine residue
Then Zmpste24 cleaves the final three amino acids off
The new final amino acid, a cysteine residue, is then methylated
It is then translocated to the nuclear envelope
Then 15 amino acids, including the modified cysteine residue are cleaved, again by Zmpste24, generating the mature lamin |
|
|
Term
| What sstructures do nuclear pores have on the nucleus side and cytoplasm side? |
|
Definition
| Nuclear pores have basket structure on the nucleus side, and spokes emanating into the cytoplasm |
|
|
Term
| What do nuclear transport proteins contain? |
|
Definition
| import signals (nuclear localisation signal NLS) |
|
|
Term
| What is the nuclear localisation signal? |
|
Definition
| Short amino acid sequence often R or K rich in one cluster e.g. PKKKRKV or bipartite with K/R rich sequences separated by about 10 amino acids e.g. KR PAATKKAGQA KKKK |
|
|
Term
| What is the nuclear export signal? |
|
Definition
| Nuclear export signal (NES) NES = Four spaced hydrophobic amino acids e.g. L as in LXXXLXXLXL |
|
|
Term
|
Definition
Transport is driven by the presence of Ran-GDP (guanosine diphosphate) in the cytoplasm and Ran-GTP (guanosine triphosphate) in the nucleus
Ran is a 25 Kda protein that can exist in a GTP bound form (“active) and a GDP bound form (“inactive”). Important roles in nuclear transport and in the formation of the mitotic spindle.
Importin binds to the NLS in order to aid passing through the pore, then Ran-GTP binds to the importin in order for it to dissociate from the protein. The Ran-GTP then passes back through the nuclear pore into the cytoplasm, and dissociates from the importin. This process requires a phosphate, turning the Ran-GTP into Ran-GDP |
|
|
Term
|
Definition
requires a phosphate, turning the Ran-GTP into Ran-GDP
To transport mRNAs out of the nucleus, nuclear export transporter 1 (NXT1) and nuclear export factor 1 (NXF1) bind to the mRNA, specifically the REF and SR sequences. CBC (Cap binding complex) and PABPII (PolyA binding protein II) then bind to the ends of the sequence, allowing the mRNA to travel through the nuclear pore. elF-4E, a translation initiation factor, then binds to the same end as CBC, and PABPI binds to the same end as PABPII |
|
|
Term
| What is the nuclues size dependant on? |
|
Definition
|
|
Term
| Why might nuclear import factors play an import role in the size of the cell? |
|
Definition
| the rate of import of nuclear lamina proteins |
|
|
Term
| What is chromatin, heterochromatin, euchromatin and the nucleolus? |
|
Definition
Chromatin = DNA + proteins e.g histones
Heterochromatin = tightly packed chromatin
Euchromatin = loosely packed chromatin
The nucleolus = site of ribosomal RNA (rRNA) transcription and ribosome assembly |
|
|
Term
| What is a chromosomes defined space? |
|
Definition
| Within the nucleus each chromosome occupies a defined space – chromosome territory. Territories are not absolute – some genes at the edge of a territory can loop out, contacts can occur between chromosomes in different territories. |
|
|
Term
| What is the nuclear matrix/scaffold? |
|
Definition
Proposed to be a network of protein and RNA rich fibres that extends through the nucleus and provides a framework for nuclear processes
Structure remaining after extraction of nuclei with high salt and treatment with Dnase I
This is controversial. Severe treatments needed to allow it to be seen – is it a real structure under physiological conditions or an artefact?
Genome organisation - loops of chromatin (DNA + histones) attached at their bases to the matrix by specific A/T rich DNA sequences called matrix associated regions (MARS)
Held in position by attachment proteins e.g. Human SAF-A (scaffold attachment factor A) |
|
|
Term
| How is the nucleus organised? |
|
Definition
Expressed genes are usually found in the interior of the nucleus, or near the nuclear pores, silent genes at the nuclear periphery
Expressed genes may be localised at the edge of chromosome territories within “transcription factories”
Transcription factories – 45-100 nm in diameter Between a few hundred and a few thousand per nucleus
Each contains approx eight molecules of RNA polymerase II and multiple active genes
Regions at the nuclear periphery are LADs (lamin associated domains) contain few active genes or epigenetic markers of gene activity
Tethering an active gene to the nuclear envelope usually turns it off
During cell differentiation many genes move between the exterior and the interior as they turn on and off
In embryonic stem cells an artificial activator which turns genes on changes their chromatin structure and leads to their repositioning to the interior |
|
|
Term
| How do we study structures in the nucleus? |
|
Definition
Fluorescent antibodies (immunofluorescence)
Uses an antibody with a fluorophore attached to recognise a specific protein (labelled primary antibody) OR uses an unlabelled antibody against a specific protein and another fluorescent antibody (secondary antibody) that recognises the first antibody
Proteins with fluorescent tags (live cells)
Green fluorescent proteins (GFP) and red fluorescent protein (RFP) widely used as tags
Both used in conjunction with fluorescence microscopy |
|
|
Term
|
Definition
Fluorescence after photobleaching (FRAP)
Express a protein with a fluorescent tag in the nucleus of target cells
Observe fluorescence within the sample
Photobleach a small area using a powerful lase
This generates a hole in the fluorescent zone
Measure the time taken for the hole to fill
This shows the mobility of the fluorescent |
|
|
Term
|
Definition
| They are involved in the modification and assembly of small nuclear ribonucleo proteins, there are about 2-3 per nucleus. Contain telomerase components, may be the site of assembly of telomerase. |
|
|
Term
| What are histone locus bodies involved in? |
|
Definition
| transcription and processing of histone mRNA. |
|
|
Term
|
Definition
PML bodies – 10-30 per mammalian cell nucleus associated with the nuclear matrix, major component is a protein called PML (promyelocytic leukemia protein) which is required for their formation
Enriched for SUMO 1 (ubiquitin-like modifier) which is covalently linked to PML. Site of SUMO modification of transcriptional activators which usually inhibits their function.
May be key sites of modification and assembly of DNA repair and apoptosis factors
P53 is phosphorylated and acetylated in PML bodies in response to DNA damage
Pml knockout mice do not form PML bodies but live normally. |
|
|
Term
| How is mRNA transported out of the nucleus? |
|
Definition
NXT1 (Nuclear export transporter 1) and NXF1 (Nuclear export factor 1) bind to the SR(which binds to the splicing enhancer sequence in the exon) and the REF (Which bind the boundary between two exons).
The cap binding complex (CBC) then binds to the SR, and the polyA binding protein II (PABPII)binds to a series of A residues
After it passes through the nuclear pore, elf-4E (a translation initiation factor) binds to the SR, and PABPI binds to the A residues |
|
|
Term
| What are lamin associated domains? |
|
Definition
| Regions at the nuclear periphery with few active genes? |
|
|
Term
| What does tethering an active gene to the nuclear membrane normally do? |
|
Definition
|
|
Term
| What are the four stages of the cell cycle? |
|
Definition
|
|
Term
| What is the difference between a closed and open mitosis, and which species do which? |
|
Definition
Lower eukaryotes such as yeast have a close mitosis. The nuclear envelope stays intact and the spindle is formed from spindle pole bodies associated with the nuclear membrane
Higher eukaryotes have an open mitosis, the nuclear envelope breaks down and the spindle is formed from centrioles which move from the centrosome to opposite poles of the cell |
|
|
Term
| How does closed mitosis occur? |
|
Definition
The spindle pole body (spb) is duplicated then one spb moves to each end of the nucleus
- Microtubules are then formed into the new bud and attach to the actin cytoskeleton
- The nucleus moves to the bud neck and the mitotic spindle forms. As mitosis is completed, the nucleus is pulled apart and one set of chromosomes is pulled into each of the cells |
|
|
Term
| What occurs to the nucleolus after mitosis? |
|
Definition
| Multiple nucleoli form around rRNA genes, then fuse together |
|
|
Term
| Which regions are in the nucleolus, and what do they do? |
|
Definition
| The centre of the nucleolus contains fibrillar regions (f) enclosed by dense fibrillar components (d) all within a granular component (g). Transcription of rRNA appears to initiate within f regions. rRNA is cleaved and processed in d regions and assembled into ribosomes in d and g. |
|
|
Term
| What are snoRNAs and what do they do? |
|
Definition
| These play a key role in rRNA processing – cleavage reactions and chemical modification of bases in the rRNA. snoRNAs can act as guide RNAs: target conversion of uridine to pseudouridine at specific positions in rRNA, or target methylation of 2’ carbon ribose sugars at specific sites. snoRNAs associate with proteins to form snoRNPs eg. the H/ACA snoRNAs associate with four core proteins – Dyskerin, GAR1, NHP2 and NOP10. H/ACA snoRNA pairs with the rRNA via sequences in each bulge. The uridines converted to pseudouridines are unpaired and lie at the base of each stem loop structure. Vertebrates – snoRNAs such as U3 involved in cleavage of rRNA are encoded by individual genes with their own promoters, transcribed by RNA pol II. Vertebrates – guide snoRNAs involved in rRNA modification are often encoded within introns of protein coding genes – the proteins are themselves involved in ribosome synthesis or the translation process |
|
|
Term
| How are ribosomes assembled? |
|
Definition
1) rRNAs are transcribed and processed in the nucleolus
2) The protein components are translated in the cytoplasm then imported into the nucleus to be assembled into ribosomal sub units at the nucleolus
3) Vertebrates – snoRNAs such as U3 involved in cleavage of rRNA are encoded by individual genes with their own promoters, transcribed by RNA pol II. Vertebrates – guide snoRNAs involved in rRNA modification are often encoded within introns of protein coding genes – the proteins are themselves involved in ribosome synthesis or the translation process |
|
|
Term
| Where are ribosomal proteins made before the ribosome is assembled? |
|
Definition
|
|
Term
| What is a proteins native state? |
|
Definition
| the most thermodynamically stable state |
|
|
Term
| In what conditions will a denatured protein refold? |
|
Definition
| Low temperature, high dilution, small proteins |
|
|
Term
| How do partially folded and misfolded proteins normally form> |
|
Definition
| The energy barrier for another fold is too high |
|
|
Term
| What would cause upregulation of heat shock proteins? |
|
Definition
Different stress conditions up-regulate hsps:
– extremes of temperature
– oxidative conditions
– expression of foreign proteins e.g. viral
– exposure to certain chemicals
All these conditions could result in damage to protein structure |
|
|
Term
|
Definition
| Preventing inappropriate interactions between complimentary surfaces |
|
|
Term
| What activities do chaperones do? |
|
Definition
‘Assisted folding’ – Conformation of target still determined by sequence
Often recognise exposed hydrophobic residues – Precise sequence varies, may involve co-chaperones
ATPase activity – ATP hydrolysis associated with conformational change – ATP binding releases polypeptide
Cycles of polypeptide binding and release – Until polypeptide folds, or is passed on |
|
|
Term
|
Definition
Exposed hydrophobic stretches on emerging polypeptide recognised by co-chaperone e.g. hsp40
Hsp40 (DNAJ) recruits hsc70/ATP, polypeptide bound to hsc70
ATP hydrolysis, promoted by hsp40, closes cleft on hsc70 (complex stabilised by Hip)
Exchange of ATP for ADP, promoted by a nucleotide exchange factor (NEF) e.g Bag protein, hsp110, releases polypeptide
Polypeptide may re-bind, cycles of binding and release possible |
|
|
Term
| How does HSP70 switch agginity states? |
|
Definition
ATP binding and hydrolysis switches HSP70 between low and high -affinity states for unfolded and partially folded protein.
Unfolded and partially folded substrate is delivered to ATP - bound HSP70 by one of several HSP40 cofactors.
ATP hydrolysis, accelerated by HSP40, closes the α - helical lid of the peptide - binding domain (yellow) and tight binding of substrate by HSP70
Dissociation of ADP, catalysed by nucleotide - exchange factors (NEFs) is required for recycling.
Opening of the α-helical lid, induced by ATP binding, results in substrate release.
Folding is promoted and aggregation is prevented when rate of the folding is greater than chaperonere re - binding or aggregation rate |
|
|
Term
| What types of ring chaperones are there, and what is their role? |
|
Definition
Large structures form double-donut rings – “Anfinsen cage” for polypetides to fold
Type I: “Chaperonin” found in bacteria and eukaryotic mitochondria and chloroplast – 7 membered rings of identical subunits – Eukaryotes Hsp/cpn 60, E. coli GroEL
Type II: found in eukaryotic and archaeabacterial cytosol – 8 membered rings – Archeasome, TRiC/CCT
Role of ring chaperones
GroEL and TRiC accept some newly synthesised polypeptides from DNAK or hsc70 – Only a subset of polypeptides – TRiC very specific for certain polypeptides
Hsp60/cpn60 accepts polypeptides newly imported into mitochndrion – Import assisted by mitochondrial hsc70 |
|
|
Term
| What do type 1 chaperonins do? |
|
Definition
Most studies use GroEL
Ring of hydrophobic residues lining upper part of cavities binds non-native polypeptides
ATP binding in cis – movement of hydrophobic residues of chaperonin away from polypeptide which is released into cavity – binding of GroES cap results in expansion of cavity to form folding cage with hydrophilic lining – ATP hydrolysis
ATP binding in trans releases GroES and polypeptide |
|
|
Term
|
Definition
| specific for certain soluble receptors and kinases |
|
|
Term
| What does prefoldin/GimC do? |
|
Definition
binds newly synthesised polypeptides and passes them to TRiC - especially actin and tubulin
TF and GimC do not have ATPase activity Scope of “assisted folding” of newly synthesized bacterial polypeptides |
|
|
Term
| What size limits are there on GroEL? |
|
Definition
Small (25kDa) polypeptides fold quickly without GroEL
Larger polypeptides (>25kDa) require GroEL
Size limit of 55kD for polypeptides in GroEL cavity – About 10-15% of total newly synthesised soluble polypeptides associate with GroEL Scope of “assisted folding” of newly synthesized eukaryotic polypeptides
Polypeptides tend to fold in domains cotranslationally – assisted by hsc70 – non-contiguous sequences of domains kept unfolded by hsc70 during translation and released to TRiC for posttranslational folding
Larger, often multi-domain, polypeptides (average ~53kD) – many of these will not fit into TriC – TriC at much lower concentration in cytosol compared to GroEL, fewer polypeptides require TriC |
|
|
Term
| What are the general principles of protein compartmentalisation? |
|
Definition
• Sorting signal(s) on polypeptide required
• Folding/unfolding – events may be involved
• Recognition factors – for sorting signal
• Translocation – machinery required to cross membrane
• Energy required – compartmentalisation increases order, decreases entropy
• Processing – sorting signals may be removed after translocation |
|
|
Term
| What types of sorting signals are there? |
|
Definition
Signals in polypeptide targeting it to correct cell compartment (or secretion)
N-terminal – direct translocation across membranes, cleaved off following translocation by signal peptidases
Internal – direct import into nucleus, peroxisomes – stop signals for membrane proteins, direct to suborganellar compartments
Recognition factors – Needed to recruit polypeptides to be compartmentalised |
|
|
Term
| How does translocation into the mitochondria work? |
|
Definition
• Most mitochondrial proteins are synthesised in the cytosol
• Kept partially folded in cytosol by chaperones (ATP hydrolysis)
• Recognised by TOM complex
• Multiple signals direct to sub-compartment
• Energy from ATP hydrolysis, proton gradient
• Chaperone-mediated folding events |
|
|
Term
| How does translocation into the ER work? |
|
Definition
• Emerging signal sequence recognised by Signal Recognition Particle (SRP) – 7S RNA plus
• The ribosome-nascent chain-SRP complex (RNC) is targeted to the rough ER by the SRP receptor: – SR: peripheral membrane component; SR: transmembrane component – SR has GTPase activity, binding of RNCSRP triggers GTPase of SRP-R and SRP – RNC released to translocon
Resting translocon closed and sealed on lumenal side by BiP (Binding Protein, ER hsc70)
• RNC binds, release of SRP permits elongation, when RNC ~70aa pore opens, then BiP released
• Elongation of NC drives translocation through pore |
|
|
Term
| How are enzymes folded in the ER? |
|
Definition
• BiP (ER hsc70/GRP78) may bind polypeptides entering ER, released when folded
• Calnexin and calreticulin may bind glycosylated polpeptides and aid proper glycosylation and folding of glycosylated proteins
• Glucosyltransferase adds glucose back on oligosaccharide chains of mis-folded proteins
• Mis-folded proteins exported from ER into cytosol and degraded by ubiquitin-proteasome system (ERAD) |
|
|
Term
| How is ubiquitin activated? |
|
Definition
• Requires E1 enzyme, ATP and Mg2+ for activation of ubiquitin with the release of PPi and AMP and formation of thiol ester bond between cysteine residue and C-terminal glycine of Ub. • Ub transferred from E1 to a member of the ubiquitin-conjugating (E2) family of enzymes to form thiol ester
• Some E2s then transfer UB directly to target protein to form an isopeptide bond between Cterminus of Ub and side chain amino group of a lysine residue in target protein
• In most cases additional ubiquitin-ligase (E3) is required to bring target protein and E2-Ub together |
|
|
Term
| How does ubiquitylation take place> |
|
Definition
| • Polyubiquitin chains can be built up on target proteins – Isopeptide bond between C-terminus Ub and lysine side chain of previous Ub – Isopeptide bonds possible between various Ub lysine residue – generates different “signals” – Lys48 chains >4 Ub lead to degradation of target by the proteasome |
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Term
|
Definition
• Present in eukaryotes, archaea and some eubacteria.
• Comprises ~ 0.5% - 2% of cellular protein.
• Two main sub-complexes, 19S & 20S, giving rise to overall 26S complex.
• Found in cytosolic and nuclear compartments.
• ATP-dependent protease.
• M.Wt ≥ 2 MDa comprising ≥ 60 subunits!
• Responsible for the degradation of ubiquitylated substrates
• Threonine protease |
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Term
| What are the targets if ubiquitylation? |
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Definition
| • Short-lived proteins – Protein half-lives vary from minutes to days – Short-lived proteins are often regulatory e.g. cyclins (control cell cycle) – Ubiquitylation may be conditional on prior phosphorylation, in turn regulated by “signals” – Intrinsically short-lived proteins contain a sequence targeting it for ubiquitylation (“degron”) |
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Term
| What is autphagy and how does it occur? |
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Definition
• The other main pathway for protein degradation is autophagy (“self-eating”)
• Vesicular pathway that delivers proteins to lysosomes for degradation
• “Nucleation” – formation of a de novo membrane in the cytoplasm that sequesters cytoplasmic components in autophagosome
• Fusion with lysosome to form autolysosome
• Low levels of constitutive autophagy produces general protein turnover
• Increased during nutrient deprivation and in response to certain signals e.g. lack of growth factors
• Involves ATG protein factors and protein modification (cf: ubiquitylation)
• Some ubiquitylated proteins (non K48) are degraded by autophagy e.g. mitophagy (autophagy of mitochondria) triggered by ubiquitylation of OMM |
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Term
| How is the balance between synthesis and degredation regulated? |
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Definition
| • Balance between protein synthesis and degradation regulated e.g. – Insulin/glucagon – change sin response to availability of nutrients – Growth factors increase synthesis and decrease degradation – Direct effect of metabolites (ketone bodies decrease muscle protein degradation) |
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Term
| Nonelectrolytes contain what type of bonds and why? |
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Definition
| covalent, to prevent dissociation |
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Term
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Definition
| Cell membrane potential, cell volume, pH buffering |
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Term
| What are HCO3, Cl- and Ca2+ involved in? |
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Definition
HCO3- is involved in pH buffering of CO2
Cl- is involved in osmotic pressure and signal propagation
Ca2+ is involved in blood clotting, nerve transmission and enzyme activity. |
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Term
| Where is the water in the body? |
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Definition
| ⅓ of the water in the body is extracellular, split ¾ in the interstitial fluid, and ¼ in the plasma. The rest is intracellular. |
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Term
| What restricts ion fluxes? |
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Definition
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Term
| What does movement of a charged solute across a membrane depend on? |
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Definition
Movement of a charged solute across a membrane depends on the chemical gradient and electrical gradient (Vm) - the electrochemcial gradient
When chemical gradient & electrical gradient cancel each other - no net flow of ion - the voltage gradient at this point is the ‘equlibrium potential’ |
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Term
| What gauges the equilibrium potential? |
|
Definition
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Term
| What is the nernst equation |
|
Definition
[image]
Where V is the nernst potential (in millivolts)
R is the universal gas constant - 8.314J.K-1. mol-1
T is the temperature in kelvin (temp in C + 273.15)
z is the valence of the ionic species e.g. +1 for Na+1
F is the Faraday constant, 96,485 C. mol-1
Xout is the concentration of ion in extracellular fluid, Xin is the intracellular |
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Term
| What is the goldmann field equation/ |
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Definition
[image]
Where pK is the relative membrane permeability for K+
pNA is the relative membrane permeability for Na+ and pCl is the relative membrane permeability for Cl−1 . The electrical gradient & concentration gradient can be utilised to do biological work - e.g. drive the transport of ions or solutes |
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Term
| What mus a polar solute do to pass through the lipid bilayer? |
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Definition
| To pass through the lipid bilayer, a polar solute must shed its hydration shell then diffuse 3nm through hydrophobic lipid before rehydration. The intermediate state has a very high activation energy and membrane proteins help lower this. |
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Term
| What three different types of ports are there, and what do they do? |
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Definition
Uniport - one molecule of one type through
Symport - one molecule and one co-transported ion both going the same way
Antiport- one molecule going in, one ion coming out |
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Term
| What ions do ports normally use? |
|
Definition
Uniports tend to be Ca2+ activated and electrogenic
Symports tend to use pi- and H+ and are electroneutral
Antiports use H+ and Na+ and are electroneutral |
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Term
| What are the differences between transporters and channels? |
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Definition
| Transporters are saturable and highly specific, channels are less specific, the transport rate is far greater, but movement direction is dependent on ion charge and electrochemical gradient |
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Term
| What do ion channels consist of and how do they open? |
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Definition
Ion channels consist of proteins that penetrate the cell membrane - Form a permeable pore - Lined with hydrophilic amino acid residues - α-helices are either domains on 1 polypeptide or found on several within a multi-protein complex
Opening (‘Gating’) - Transmembrane domains twist like an iris - A distinction can be made based on the cause of gating: |
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Term
| What is a permeability loop? |
|
Definition
| Voltage-gated channels contain a short P- (permeability loop) that acts as a selectivity filter - Competes with water for ions - Strips hydrate cover |
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Term
| What the sodium potassium pump do? |
|
Definition
| Na+ ,K+ Pump - located in the plasma membrane - actively pumps: - 3 Na+ from the cytoplasm → Extracellular fluid - 2 K+ from the Extracellular fluid → cytoplasm - maintains a Na+ ,K+ gradient across the cell membrane |
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Term
| What must active transport pumps be coupled to? |
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Definition
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Term
| What is important about the sodium glucose co transporter, and how does it work? |
|
Definition
Na+ , glucose co-transporter:
- Na+ moves down its concentration gradient into cell due to gradient created by Na+ ,K+
-ATPase located at cell basal surface
- SGLT (Na+ glucose transport protein): At the apical surface glucose is taken into the cell with Na+ down their concentration gradient by facilitated diffusion.
- SGLT are important for reabsorbing glucose in kidney
One of the most versatile of cell signals, it is involved in controlling most cell physiological processes - cells keep cytoplasmic Ca2+ levels 4 x lower than the ECF - gating of channels causes influx - binds effector proteins leading to output signals |
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Term
| In which ways do cells modulate Ca2+? |
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Definition
| - Intracellular localisation - Intensity (amplitude) - Temporal fluctuation (frequency) - Develops stationary & spreading waves |
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Term
| How is calcium transported against the concentration gradient into the mitochondria? |
|
Definition
| A uniport that only works at high concentrations and uses a charge gradient geneated by ETC |
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Term
| Why do voltage controlled ca channels undergo autoinactivation and how? |
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Definition
| To prevent fatal release of calcium, and the alpha subunit plugs the pore |
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Term
| What prolongs gating of voltage controlled ca channels? |
|
Definition
| phosphorylation by PKA or Ca2+/calmodulin dependent protein kinase II |
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|
Term
| What are the four main ways of cell signalling? |
|
Definition
Contact dependant - membrane bound signalling molecules contact when cells are close enough
Paracrine - signalling molecule released into local extracellular space
synaptic - Neurotransmitters and neurons
Endocrine - hormones released into bloodstream |
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Term
| What are the "first messengers"? |
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Definition
| hormones, growth factors, cytokines |
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Term
| How does paracrine signalling effect the cell? |
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Definition
| Signal molecule binds to receptor - then either enters nucleus and alters protein synthesis, or alters protein function via an intracellular signalling pathway |
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Term
| What types of cell surfaace receptors are there and how do they work? |
|
Definition
Ion coupled channels- signal molecule binds, channel opens or closes
G-protein coupled receptors - signal binds to receptor, g protein activated, g protein activates an enzyme
Enzyme coupled receptors - signal molecule binds to receptor, which either forms a dimer and becomes an enzyme, or activates an associated enzyme |
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Term
| In what ways do signals passed from receptor to intracellular proteins cause change? |
|
Definition
– direct protein-protein interactions
– covalent modification e.g. phosphorylation/dephosphorylation
– binding of small molecules/ions (e.g. GTP, cAMP, calcium) some of which are “second messengers” whose concentration varies depending on “first messenger” (ligand/hormone) binding to receptor |
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Term
| How does signalling via phosphorylation alter things? |
|
Definition
| Turns on or off an enzyme normally |
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|
Term
| How does signalling via GTP-binding work? |
|
Definition
GTP binding and GTP hydrolysis by G-proteins alters their conformation, activating a switch
GTP binding to monomeric G-proteins (e.g. Ras) stimulated by Guanine nucleotide Exchange Factors (GEF), GTPase activity stimulated by GTPase Activating Proteins (GAP)
Ligand activated GPCRs stimulate GTP binding to trimeric Gproteins (act as GEF), effector enzymes stimulate (act as GAP) GTPase |
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Term
|
Definition
Signal transduction often amplifies the original signal
– hormone concentrations typically ~ 10-8M
– receptor has high affinity for ligand
– ligand-receptor complex activates many intracellular proteins
production of “second messengers” at higher concentration than first messenger
cascade of signalling protein activation amplifies signal e.g. phosphorylation cascades |
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Term
|
Definition
Different signal transduction pathways (from different first messengers) may converge on a common target – To enhance the response or to attenuate the response
Different extracellular signals (ligands) acting via different receptors on the same target cell may regulate the same downstream signals (cross talk). Final cellular response will depend on combination of signals arriving at cell surface. |
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Term
| What are transient signals? |
|
Definition
Intracellular signalling systems must be switched on and off:
– Switched on by receptor activation
– Switched off when receptor unoccupied
– G-proteins hydrolyse GTP, GDP - G protein inactive
– phosphorylation state of proteins returned to the resting state by phosphatases or kinases
– second messenger molecules degraded or sequestered – conformational state of proteins returns to resting state |
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Term
| What can prolonged exposure to extracellular signals lead to? |
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Definition
| decreased sensitivity to the signal |
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|
Term
| What is the pathway that stems from adrenaline? |
|
Definition
| Adrenaline binds to beta-adrenergic receptor, effector is adenylate cyclase, second messenger is cAMP, causes glycogen breakdown |
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Term
| What is the pathway that stems from Light? |
|
Definition
| rhodopsin receptor activated, effector is cGMP phosphodiesterase, second messenger is cGMP, causes photoreception |
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|
Term
| What is the pathway that stems from fMet peptide? |
|
Definition
| fMet peptide binds to chemotactic receptor, effector is phospholipase C, second messenger is IP3, causes chemotaxis |
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Term
| What is the pathway that stems from acetylcholine? |
|
Definition
| Acetylcholine binds to muscarinic Ach receptor, effector is K+ channel, second messenger is K+, slows pacemaker activity |
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Term
| G protein coupled receptors often have a... motif |
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Definition
|
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Term
| What do heterotrimeric g-proteins do? |
|
Definition
| Heterotrimeric G-proteins interact with cytoplasmic loops and cytoplasmic faces of transmembrane domains of GPCRs • Heterotrimeric G-proteins are intermediaries between receptor and second messenger production • Ligand binding to receptor triggers change in heterotrimeric G-protein • G-protein “senses” receptor occupancy |
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Term
|
Definition
| G proteins “sense” receptor state (occupied/unoccupied) • Occupied receptor acts as GEF – promotes GTP exchange for GDP on Ga • Ga usually dissociates from Gbg • Ga and/or Gbg activate/inactivate effector enzymes which alter concentration of 2nd messengers |
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Term
| What is the epinephrine pathway? |
|
Definition
binds to receptor
causes replcaement of GDP by GTP, activating G protein, G protein activates adenylyl cyclase, which catalyzes formation of cAMP, which is either degraded, or activates PKA, which phosphorylates some proteins. Id say which ones but the module doesnt bother to mention that. |
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Term
| Which ligands increase cAMP? |
|
Definition
| Glucagon, adrenaline, vasopressin, ACTH, thyrotropin |
|
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Term
| Which ligands decrease cAMP |
|
Definition
| Adrenaline, adenosine, acetylcholine, somatostatin |
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Term
| How does cAMP bind to sensors? |
|
Definition
– Protein kinase A PKA activation by cAMP
• 2 cAMP molecules bind per R subunit – affinity of R for C decreases 103 -105 fold on cAMP binding
• PKA dissociates into a dimer of R(egulatory, inhibitory) and two C(atalytic) monomers
• Inhibition released, catalytic subunits active, phosphorylates target proteins
Regulatory subunits bound to catalytic subunits via “pseudosubstrate” sites +4 cAMP 2 cAMP bind to each R sub-unit Pseudo-substrate site on R buried Catalytic sub-units released and active |
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Term
| How does densitisation work? |
|
Definition
| Specific receptor kinases also phosphorylate receptors – prolonged activation of receptor activates GPCR Kinases (GRKs 1-6) – GRK1 also know as rhodopsin kinase, GRK2 as bARK1 – phosphorylate 7 serine/threonine residues on occupied receptor – promotes binding of arrestin to receptor Cterminus, Ga binding blocked - homologous desensitisation |
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Term
| How does receptor recycling work? |
|
Definition
| Receptor re-cycling • Prolonged activation leads to internalisation of receptors • May be triggered by phosphorylation and arrestin binding • Arrestin may recruit clathrin components for endocytosis • Ligand released in acidic endosome, receptor dephosphorylated - “reset” mechanism • The more recycling the greater the chance of receptor lysosomal degradation – down-regulation |
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Term
| How does calcium signalling work? |
|
Definition
| Mobilisation of calcium involves a water soluble product of phosphatidylinositol bis phosphate (PIP2 ), inositol 1,4,5 tris phosphate (IP3 ) which release Ca 2+ from intracellular stores • Calcium then alters the activity of calciumdependent enzymes Phosphoinositides • Phosphatidylinositol 4,5 bis phosphate (PIP2 ) is a phospholipid found in small quantities in cell membranes • PIP2 is a polyphosphoinositide produced by phosphorylation of phosphatidyinositol • PIP2 is hydrolysed by PI specific phospholipase C to release IP3 and diacylglycerol (DAG) • There are other polyphosphoinositides important in other signalling pathways |
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Term
| How does calcium regulate itself? |
|
Definition
| high Ca2+ prevents IP3 binding, calmodulin binds, closes pore – calcium spike |
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Term
| What is calmodulin dependent protein kinase II? |
|
Definition
| Calmodulin-dependent protein kinase II – has broad specificity – Phosphorylates synapsin-1 which frees neurotransmitter granules for exocytosis – Phosphorylates and activates enzymes involved in neurotransmitter synthesis – Calmodulin-Ca2+ partially activates CaM Kinase-II which then autophosporylates to fully activate form – CaM Kinase II remains active when Ca2+ drops, until dephosphorylated – “memory” of elevated Ca2+ |
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Term
| What is protein kinase C? |
|
Definition
| Protein kinase C (PKC) • A calcium dependent kinase found in soluble form which associates with the plasma membrane • Plasma membrane association promoted by diacylglycerol (DAG) released by PLC • In the presence of DAG, PKC active at lower calcium concentrations • Calcium and DAG will act synergistically to activate PKC |
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Term
| What does vasopressin do in the liver? |
|
Definition
| Vasopressin stimulates glycogenolysis in liver (V1 receptor) – released by the neurohypophysis of the pituitary, also regulates blood pressure and volume – Activates PLC which produces IP3 and DAG – IP3 triggers release of intracellular Ca2+ stores • Ca2+ activates calmodulin-dependent protein kinase which phosphorylates glycogen synthase (inactivates) and phosphorylase kinase (activates) |
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Term
| What are receptor tyrosine kinases? |
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Definition
| RTKs are single pass membrane proteins • Binding of ligand leads to dimerisation and activation of the tyrosine kinase activity • Some ligands are soluble, others are membrane associated e.g ephrins • The RTK auto-phosphorylates on the cytosolic side • Phosphorylation leads to recruitment of proteins with SH2 (majority) or PTB domains |
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Term
| Which protein will dock with RTKs? |
|
Definition
| Phospholipase Cg - leading to an increase in [Ca 2+] i – Cytoplasmic tyrosine kinase Src which phosphorylates cytosolic proteins – Phosphoinositide 3-kinase which phosphorylates lipids to create docking rafts at the plasma membrane – Many of these docking proteins have SH£ domains which allow them to bind other signalling factors with proline rich motifs |
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Term
| How do RTKs regulate monomeric G proteins? |
|
Definition
| RTKs (indirectly) regulate monomer4ic Gproteins via GEFs • Ras is a monomeric G-protein regulated by GEFs and GAPs • Ras-GEFs can act as a scaffold for downstream targets of Ras • Often involved in regulation of gene expression in cell proliferation of differentiation • Mutations in Ras lead to cancer |
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Term
| What activates the MAP kinase cascade? |
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Definition
| Ras activates the Mitogen Activated Protein kinase cascade • Takes the signal into the nucleus – gene regulatory proteins phosphorylated • Transient signal – positive and feedback loops determine duration of response • “Intermediate early genes” targeted by MAP kinase (Erk) |
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Term
| How does cytokine signalling work? |
|
Definition
Cytokine signalling • Cytokines (local mediators) and some growth factors (Growth Hormone) act through receptors linked to Janus (tyrosine) Kinases (JAKs) • JAKs phosphorylate STATs (Signal Transducers and Activators of Transcription) which migrate into the nucleus and alter transcription of some genes
Cytokine Signalling • Occupied receptors bring two JAK molecules together, activated they phosphorylate each other and then the receptor • Phosphorylated receptor recruits SATS via their SH2 domains allowing phosphorylation of the SAT by JAK • Phosphorylation releases STATs which form dimers via SH2 domains and migrate into nucleus and activate transcription of some genes • Some gene products are inhibitory proteins that terminate the signal (negative feedback) |
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|
Term
| Why do cells need vesicular transport? |
|
Definition
| To eat, commyunicate, regulate protein delivery and coordinate protein syntheis, modification and delivery |
|
|
Term
| What types of proteins are constitutive secretory? |
|
Definition
|
|
Term
| What types of proteins are regulated secretory? |
|
Definition
| peptides, digestive enzymes, milk proteins |
|
|
Term
| What does Brefeldin A do? |
|
Definition
| It has a broad effect on aneterograde movement but les on retrograde |
|
|
Term
|
Definition
| disrupts microtubules in vesicular tubular clusters |
|
|
Term
| What three types of coated vesicles do we know of? |
|
Definition
|
|
Term
| Wgat do we know about the clathrin coat? |
|
Definition
| Clathrin composed as a triskelion. – 3 heavy chains (~180kDa) – 3 light chains (~35 - 40kDa) • AP (adaptin) composed of a heterotetramer. – α, β, γ and µ subunits |
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Term
| What do we know about COPI? |
|
Definition
| COPI is a ~700kDa ‘coatamer’ complex comprising: – COPα,β,β’,γ,δ,ε and ζ proteins. – β-subunit shows some homology with adaptins • COPII composed of two sub-complexes: – Sec23/24 tetramer – Sec13/31 complex • No sequence homology between the COPI and COPII complexes. |
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Term
| What controls coat assembly? |
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Definition
| Monomeric GTPases control coat assembly • Membrane transport to and from an organelle must be balanced. • Coat assembly must occur only when and where needed. • Controlled by monomeric coat-recruitment GTPases: – ARF controls coat recruitment to clathrin and COPI vesicles – Sar1 performs the same function for COPII vesicles • Coat-recruitment [GTPases] usually high in cytosol as GDP-bound state. • Process initiated by interaction of GTPase with membrane-bound GEF. |
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Term
|
Definition
| SNARE characteristics • At least 20 SNAREs in an animal cell. • Comprised of v-SNARE (vesicle) and t-SNARE (target). • Contain characteristic helical domains. • Helices of v-SNARE and t-SNARE interact and intertwine to form stable trans-SNARE-complex. • Trans-SNARE-complexes always contain a characteristic 4-helix bundle. SNAREs may mediate membrane fusion • Once docking has occurred, fusion does not always occur immediately. • Docking and fusion are two separable processes. • Fusion requires close approach of the two membranes. • SNAREs may behave a bit like a winch to draw the membranes together. Interacting SNAREs must be separated before reuse • Most SNAREs have already been used for targeting. • SNAREs must be separated |
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Term
| What are Rab proteins needed for? |
|
Definition
| Rab proteins are essential for accurately targeted vesicle docking • SNAREs require cooperation with Rab proteins for accurate vesicle docking. • Rab proteins comprise ~60 known family members. • Monomeric GTPases. • Located in both cytosolic and membrane-associated fractions. • GDP-bound form is soluble and inactive. • GTP-bound form is membrane-associated and active. |
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Term
|
Definition
| After budding, ER vesicles fuse with one another. • SNARE contribution during fusion is symmetrical. • These multi-vesicular compartments are known as vesicular tubular clusters (VTCs). • VTCs differ from ER and Golgi compartments; also known as ERGIC (ER-Golgi Intermediate Compartment). • Migrate using microtubule networks. • VTCs bud COPI-coated vesicles for return of ER-resident and ER-budding proteins. |
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Term
| What is glycosylation important for? |
|
Definition
| Glycosylation is important for localization and function • The ERGIC53 protein serves to recognise high-mannose proteins. • The Notch protein requires specialized glycosylation to signal correct cell fate. • Mannose-6-phosphate controls transport into the lysosome. |
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|
Term
| What can cause lysosomal storage diseases? |
|
Definition
| GlcNAc phosphotransferase defects cause lysosomal storage disease • Lysosomal storage diseases occur if one or more hydrolases are defective. • Hurler’s Disease is caused by a defect in an enzyme involved in glycosaminoglycan turnover. • Rare, severe lysosomal storage disease known as I-cell Disease. • I-cell disease caused by defective or missing GlcNAc phosphotransferase. • All lysosomal enzymes normally acquiring M6P modification are mislocated. • Other lysosomal targeting systems occur in some cells i.e. hepatocytes |
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|
Term
| What properties does receptor-ligand binding have? |
|
Definition
| non-covalent adn reversible |
|
|
Term
| How would you assess receptor binding dynamics? |
|
Definition
Use radioactively labelled hormones
- expose cells to labeeled hormone
allow equilibration, then seperate free and bound hormones via centrifugation or filtration, analyse free vs bound hormone, and plot on graph. |
|
|
Term
| How can voltage changes be measured? |
|
Definition
| Use a large cell and an electrode - only gives an average |
|
|
Term
| How do you seperate organelles? |
|
Definition
| Disrupt cell membrane non-destructively, via sonification, fractionate componenets using ultracentrifugation |
|
|
Term
| How do you obtain pure fractions? |
|
Definition
Density gradient centrifucation
Slow drip, heaviest collected first |
|
|
Term
| How can gene expression by analysed? |
|
Definition
Conventional PCR - only gives end point descruption
Real time PCR - analyse transcription as it occurs
Oligonucleotide probes - hybridize to the target sequences, release reporter which emits fluorescence |
|
|
Term
| Northern blotting uses... |
|
Definition
|
|
Term
| Identification by mass spec |
|
Definition
| Magnettic field deflects ions based on size and charge, the lighter and more charged, the more feflected, and the remaining ion beam can be identified |
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