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BMS238 Cell and molecular > Part 4 > Flashcards

Part 4 Flashcards

1
Q

What does protein trafficking consist of?

A
  • membrane protein synthesis
  • Protein tagging by sugars (glycosylation)
  • Protein activation by trimming (proteolytic processing)
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2
Q

What do cells carry sugars on the outside?

A

Protect themselves from harsh environment and provide a tagging mechanism

  • epithelial cells rich in carbohydrates
  • provide protection from foreign elements

*Both lipids and proteins are often tagged by complex sugars

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3
Q

Different types of proteins

A
  • Cystosolic protein= made it cytosol
  • membrane and secretory proteins= made in rough EF
  • Nuclear proteins= made on outer nuclear membrane

Rough ER- Smooth ER- Golgi- secretory vesicle- plama membrane

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4
Q

Protein synthesis starts at cytosol then goes to Rough ER

A

Free ribsome cycle- mRNA encoding a cytosolic protein remain free in cytosol
Membrane bound ribosome cycle- rough ER
- ER singal sequence goes to ER membrane where mRNA encoding a protein targeted to ER remains membrane bound- Polyribsosome bound to ER by multiple nascent polypeptide chains

*Common pool of ribosomal subunits in cytosol

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5
Q

Following synthesis pattern

A

Proteins move into smooth ER (where lipids are made and vesicles form)
Then to Golgi for final addition of sugars and sorting into place
Secretory vesicles then trim (protease) and activate hormones/ enzymes during maturation of vesicles

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6
Q

Signal recognition particle

A

(SRP) guides ribosome following binding to the signal peptide

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7
Q

SRP and its receptor allow attachment of ribosome to translocator

A
  1. Binding of SRP to signal peptide causes a pause of translation
  2. SRP-bound ribosome attaches to SRP receptor in ER membrane
  3. Translation continues and translocation begins
  4. SRP and SRP receptor displaced and recycled
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8
Q

Differential centrifugation

A

Separates heavy and light vesicles

  • Rough microsomes have a high density and stop sedimenting and float at high sucrose conc
  • Smooth microsomes have a low density and stop sedimenting and float at low sucrose conc

Low= high in tube

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9
Q

What shows real topography of ribosomes on ER membrane?

A

Electron microscopy

ER-bound signal peptidase removes signal peptides from secretory proteins

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10
Q

How do transmembrane proteins stay anchored in the ER membrane?

A 
Swapping the signal peptide for a lipid anchor in the ER can take place to continue membrane association 
Glycosylphosphatidylinatol anchor (GPI anchor)
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11
Q

Protein maturation

A
  • The signal sequence is removed by a specific signal peptide
  • Followed by conformational maturation
  • Disulfide bridges are formed between cysteine residues to solidify protein shape
  • Protein is glycosylated by standard carbohydrate chain ( quality control)
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12
Q

What causes return of proteins?

A

ER residuent enzymes carry KDEL (lysine- asparatic acid glutami acid leucine) sequence important for their return
Golgi to ER

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13
Q

Addition of carbohydrates to protein is important for

A

Protein stability- in harsh environment
cell- cell communication
cross species separation

trimming and growth of carbohydrate chains proceed step by step in individual Golgi cisterine

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14
Q

Each glycosylation step requires separate Golgi compartment to keep specific glycosylation

A

Cis Golgi network- sorting phosphorylation of oligosscharides on lysosomal proteins
Cis cisterna- removal of man
Medial cisterna- removal of man and addition of GLCNA
Trans cisterna- addition of gal and NANA
Transgolgi network- Sulfation of lyrosine and carbohydrates

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15
Q

What are limitations in organ transplantation due to?

A

Simple sugar modifications
Human cells make beta glucose while other animals alpha galactose Humans will produce antibodies against alpha galactose and thus reject transplant
* Animal derived transplants are rejected by humans

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16
Q

Which animal make organ transplantation possible?

A

Genetically modified pigs lacking alpha galactose

  • Enzyme add a sugar= normal a-gal +ve pig cells attack antibodies causing hyperactive rejection
  • a-1,3 gal enzyme KO via gene targeting= a-1,3 gal neg pig cells- donor ce cells to produce cloned
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17
Q

Example of protein glycosylation within human population

A

Carbohydrates of band 3 protein in erthroyctes determine human blood compatibility
4 groups- O, A , AB, B
0-lacks terminal gal
A- acetylated gal
B- normal gal
Blood group determined by single terminal galactose residue. Terminal galactose determines blood group compatibility through immune compatibility.

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18
Q

Blood group rbc and plasma antigen

A

Type A- Antigen A and BP B (BP=blood plasma)
Type B- Antigen B and BP A
Type AB- Antigen A +B and BP neither A or B
Type 0- antigen neither A or B and BP A+B

O can give blood to any, A and B can give to AB

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19
Q

What can blood group be used to determine

A

Paternity

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20
Q

Protein trimming

A

Takes place prior to selection
Insulin example
1. preproinsulin translation, signal cleavage, proinsulin folding
2. proinsulin is transported to Golgi
3. proinsulin is cleaved to produce mature insulin and G peptide

trimming= processing/ maturation

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21
Q

Type 1 diabetes

A
  • Secreted proinsulin rather than insulin
  • Missfolding of proinsulin in ER due to a mutation
  • Protease in secretory vesicle cannot cleave off the c peptide
  • Secretion of dysfunctional pro-insulin instead of insulin into blood
  • Generation of antibodies against the pancreatic cells
  • Destruction of pancreatic cells
  • Type 1 diabetes= blood glucose increase
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22
Q

Cleavage of opiomelanocortin can give rise to several hormones

A

ACTH and B-lipotrophin- secreted by the pituitary gland

B- endorphin- generated by neurons in responses to excerise/ stress . has approx. 80x the potency of morphine

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23
Q

Protein trafficking relying on vesicular transport

A

Rough ER (protein sugars) - Smooth ER ( lipids) - Golgi (sugars) - Secretory vesicle (final trimming) - plasma membrane (secretion)

mitochondria- energy

24
Q

How is lipid synthesised at the smooth ER membrane

A

Form anchoring due to linking 2 fatty acids

Headgroup addition

25
Consituative and regulatory pathway
Constructive - Golgi- Newly synthesised soluable proteins and membrane - Unregulated membrane fusion Regulatory - Golgi- secretory vesicle storing secretory proteins - Regulated membrane fusion (intracellular signal pathway) - regulation involves accumulation and storage before signal triggered release
26
What does insulin trigger?
Delivery of glucose transporters into the plasma membrane of muscle cells
27
Insulin stimulated cell
Signal causes relocation of glucose receptor to PM to boost glucose uptake RAB protein= Identify target membranes for fusion
28
Type 2 diabetes
Dysfunctional glucose uptake into muscle and cell fat
29
What are the clinical syndromes mutations in insulin receptors leads to?
1. leprechaunism- fatal 2 years, elfin like features: protuberant ears, large hands and feet, decrease in subcutaneous fat 2. Robson- mendenhall syndrome-skin and teeth abnormalities, hair outgrowth 3. Type A insulin resistance
30
How is insulin produced?
From beta cells of islets and is secreted into blood stream - high processed after made - has to cleaved multiple times held together by disulfide bridges between cysteines
31
What is the insulin intermediate effect?
Glucose uptake from the blood into muscle cells and odipocyles
32
Long term exposure of insulin
Increased expression of liver enzymes that synthesise glycogen and of apodicytes enzymes that synthesise triacylgylcerols
33
How does insulin result in action of IRS
Insulin binds to receptor made up of 2 subunits- beta and alpha Both synthesised as a single polypeptide which is cleaved into 2 fragments - binding of insulin results in autophosphoylation - insulin receptor substrates (IRS) contains a phosphotyrosin binding domain (PTB) and is highly phosphorylated by the insulin receptor - IRS then acts as a clocking site for many other proteins, such as GRB2 to activate the RAS pathway
34
RAS pathway
Phosphorylation of kinases - MAP kinases - TF
35
RAS independent signalling
PI-3 kinase is made up of two subunits: -P85 which has an SH2 domain - P110 which is a kinase Upon binding to IRS, PI-3 kinase phosphorylates p14,5- biphosphate and PI 4 phosphate to PI3,4,5 triphosphate and PI 3,4-biphosphate come together and form complex
36
What happens once they are recruited to the membrane? RAS
Pkb is phosphorylated by membrane associated kinases 3-PKD1 (Phosphoinoside-dependent-kinase) Pkb then goes through conformational change to become active and its then released and effects numberous proteins
37
What proteins does PKb effect
- glycogen synthase kinase - glucose transporter (+) - foxo (-) - phosphoenolypyruvate carboxykinase
38
How can we identify which genes are activated by insulin signalling?
Track gene expression (with microassay) before and after adding insulin to cells in culture Sometimes we known the exact mechanism of regulation of insulin eg. when low blood insulin levels, foxo binds to an IRS near to a PEPCK activating its transcription. High insulin levels results in phosphorylation of foxo1 by PKB which inactivates it
39
How is PCR used to study amplify DNA
Polyermase chain reaction clone DNA from minute samples Quantify levels of RNA -Template/double stranded DNA- heat- separate- hybridisation of primers- DNA synthesis from primers *Separate- DNA synthesis- repeat for 3rd cycle
40
What do we design primers based on?
Target sequence
41
The most common primer
TAQ- Thermus aquaticus bacteria | Heat stable DNA polymerase
42
What are the different types of clones that can be formed from PCR
Genomic clones | lDNA clones
43
qPCR
1. make cDNA from tissue 2, perform PCR in the presence of a fluorescent DNA dye 3. measure the fluorescence after each pcr cycle
44
Promoter bashing
Make a transgene that uses a quantifiable promoter luciferase produces fluorescent substrate that is quantifiable then make a series of deletions and test the responsiveness to insulin *use in conjuction with EMSA and DNAse 1 protection
45
Promoter analysis of HIGFBP-1
Identify putative binding sites in critical region based upon sequence similarity eg. compare sequence from genes that you have found to be repressed by insulin - clone upstream of reporter - leucifine single based changes can be used to fine map the binding
46
What does the promoter binding analysis show?
1. shows a loss of understanding by dex | 2. show a reduction in activation by dex and a loss of insulin repression
47
What is Virchow about cancer (1858)
Omnis cellula e cellula- cancer arises from normal tissue cells- including tumours
48
What is hyperplasia, metaplasia, dysplasia, neoplasia?
``` Hyperplasia= tissue growth containing excessive numbers of cells Metaplasia= tissue growth displaced but otherwise normal- oesophageal section Dysplasia= tissue growth where cells appear abnormal Neoplasia= invasive abnormal tissue growth ```
49
Which is the most aggressive
Neoplasia
50
Histopathology to provide clues to trace origins of tumours
Yes deal with tissue that may be displaces- treatment strategy to find the type of cell Eg mouse lung tissue with melanoma- cancer of melanocytes= black pigment
51
Why is histopathology important?
It allows us to predict common behaviours among diverse tumours
52
Causes of cancer
nature v nurture | Genetics v environment
53
Risks in cancer
Age-1/3 get cancer in lifetime | 80% if you smoke
54
Cancer risk affected by
External factors assignable environment influences Eg depends on country- japan high stomach cancer- risk decreases if they move
55
Genetic basis for cancer
Some families are susceptible to particular cancers - Retina= retinoblastoma - Kidney = wilms tumour - Colon= adenomatous polyposis coli - Breast= hereditary breast cancer

BMS238 Cell and molecular (4 decks)

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