proteopathic diseases and amelogensis imperfecta

Question 1

what is a ‘proteopathic disease’

Answer 1

a class of diseases where proteins are STRUCTURALLY ABNORMAL due to protein misfolding –> disrupt the functions of cells/tissues

e.g. misfolding can be due to mutations= abnormal aggregation of protein

Question 2

describe the process of protein secretion/exocytosis briefly

Answer 2

mRNA generated in nucleus.

mRNA leaves nucleus and is transported via ribosome to cytoplasm (if intracellular p) or the ER (if extracellular p)

the p is synthesised on the ribosome and will fold into its 3D shape in the ER. P modifications occur.

a region of the ER buds off to form a transport vesicle that contains the modified p

the vesicles enter the Golgi apparatus, fusing with them to empty out their contents. here, enzymes will further modify the P.

P then are packaged into secretory vesicles which will fuse with the PM and the p will be released outside.

Question 3

how does the cell diffrentiate between secretory proteins that need to be translocated to the ER?

Answer 3

there are 2 mechanisms:

1. CO- TRANSLATIONAL TRANSLOCATION. Here, the n-terminal has a signal sequence which is recognised by the signal recognition particle, SRP which will trigger the translocation to ER WHILST STILL BOUND TO THE RIBOSOME.
2. POST-TRANSLATIONAL TRANSLOCATION. Here, the protein is first fully translated, separated from the ribosome, THEN the translated signal sequence is recognised by other proteins which will allow it to enter the ER.

Question 4

what are chaperone proteins?

Answer 4

a group of proteins that guide protein folding so that they do not mis-fold and aggregate within cell= cause cell/ER stress= may lead to disease

Question 5

what is the unfolded protein response/UPR ?

Answer 5

a type of quality control… p folding is a chaotic process and approx 30% of p. are misfolded/ incorrectly processed under normal situations. the UPR kicks in and controls ER STRESS caused by these misfolded proteins.

it involves 3 receptors/ ER stress sensors: IRE1, PERK, ATF6 which are bound to chaperones like BIP, in an inactive confirmation, when there is NO ER stress!

Question 6

how do chaperones and the UPR link together

Answer 6

UPR has 3 ER stress sensors: PERK, IRE1, ATF6.

ER chaperones like BIP are master REGULATORS of the UPR via direct interactions with the 3 sensors. when there is no ER stress, BIP and other chaperones are bound to the stress sensors so they are inactive. When there is a misfolded protein, Bip will bind to the misfolded protein, thus DISSOCIATE away from the ER sensors which then become ACTIVE and trigger downwards signalling known as the UPR.

Question 7

describe the structure of PERK

Answer 7

PERK is transmembrane protein thus it has an ER luminal side (which interacts with chaperone proteins) and a cytosolic side (which has a kinase domain).

Question 8

describe the mechanism of how PERK works in the UPR

what is the main mechanism by which PERK works in the UPR?

Answer 8

main function of PERK= dampen down overall protein synthesis in the cell.

it works by phosphorylating eIF2alpha = the 80s ribosome doesnt assemble properly= no translation AND it also causes selective translation of proteins which will promote ER folding.

Question 9

what is the main mechanism by which IRE1 works in the UPR?

Answer 9

when chaperones dissociate from IRE1, it DIMERISES and auto-phosphorylates itself.
it causes regulated mRNA splicing of transcription genes which increase the folding capacity of the ER

Question 10

what is the main mechanism by which ATF6 works in the UPR?

Answer 10

when the chaperone dissociates from ATF6, it is translocated from the ER to the golgi= here it is proteolytically cleaved= becomes active= induces translation of genes that increase folding capacity of the ER

Question 11

the UPR is a dynamic process and it is all about a HOMEOSTASIS between UPR activation, reducing the amount of proteins entering the ER and ERAD AND VS ER-stress induced apoptosis (when damaged cells are beyond repair)

why would apoptosis be triggered and give an example of a protein involved

Answer 11

While UPR activation leads to adaptations that may sustain cell survival… under SEVERE and PROLONGED ER stress conditions, where the cells fail to restore ER homeostasis, the UPR will ACTIVATE pathways that lead to apoptotic cell death

e.g. ER-stress induced apoptosis can be carried out via CHOP.

Question 12

what is ‘amelogensis imperfecta’

Answer 12

a GROUP of DEVELOPMENTAL CONDITIONS, which are GENOMIC IN ORIGIN, that affect the STRUCTURE and APPEARANCE of ENAMEL on nearly every tooth in an EQUAL MANNER

inheritence may be autosomal dominant, autosomal recessive or sex-linked.

Clinically, enamel may show HYPO-plasia (also may say ‘hypoplastic’) OR HYPO-mineralised or both.

teeth are sensitive, discoloured, and prone to dis-integrating after eruption.

Question 13

which genetic mutations are involved in AI?

Answer 13

enam S555I

amelx Y64H

Question 14

the enamel matrix is 90% amelogenin.

amelogenin is encoded on both the X and Y chromosome in humans.

human males=XY
human females=XX

what is distinct about the amelogenin gene on the y chromosome?

Answer 14

it transcribes only 10% of the amelogenin protein made by the x chromosome, therefore even if it not affected, if the x chromosome is affected, it is not enough to over-ride disease.

Question 15

what is the name of process when there is random inactivation of the second X chromosome in females

Answer 15

lyonisation

Question 16

what is the effect of lyonisation

Answer 16

in a heterozygous female with X-linked AI, one outcome is 50% of ameloblasts affected and the second outcome is 50% un-affected ameloblasts..this gives rise to a BANDING ENAMEL pattern seen only in females.

Question 17

in human males, what is the outcome if they have are homozygous for amelgenin gene mutation?

what if they are hetrozygous for the mutation?

Answer 17

100% of the enamel found will be affected (no enamel will be made basically)

also 100% affected in hetero male because the WT-copy from Y is not enough to overide the transcription of X

Question 18

the the EARLY phases of enamel formation, even in a mutant mouse, the affected ameloblasts which are deposited are normal giving rise to ‘normal enamel’. at which stage is this disrupted?

Answer 18

maturation phase.

this gives rise to an abnormal outer layer on the enamel which a normal layer underneath.

Question 19

outline other key findings from research done into mutant nice with AI?

Answer 19

mutants:

1. esionophillic vesicles seen which CO-LOCALISE with the amelogenin protein so they enter into the vesicles.
2. the amelogenin mutation causes loss of normal golgi and ER structures.
3. mutant amelogenin ACCUMULATES in the cell which suggests a failure in the SECRETORY pathway= causes ER stress= activates the UNFOLDED PROTEIN RESPONSE, UPR.
4. the data concluded that the amelx y64h + enam s551 mutation accumulate= ER stress= activate the UPR = initially works by responding fixing the problem to cause a NORMAL enamel layer by secreting PHENYLBUTURATE (chemical chaperone) which stops protein misfolding BUT THEN, the ER stress exceeds the UPRs ability to cope= the apoptotic pathway is favoured= results in secretion of abnormal enamel

Question 20

can phenylbuturate be used to treated AI according to study with mice?

Answer 20

yes there was marked improvement, specifically on female mice.