Molecular/cell Biology Of Mitochondrial Diseases

Question 1

How is ATP produced?

Answer 1

In a process called oxidative phosphorylation
Four enzyme complexes make up the ETC
These receive reducing equivalents (NADH and FADH2) from glycolysis, fatty acid oxidation and the citric acid cycle
These are oxidised to NAD+ and FADH by the complexes which accept the electrons and transport them down the chain
This movement of electrons down the chain (electron motive force) is converted into a proton motive force and H+ to be transported into the inter- membrane space.
The build up of H+ in the inter membrane spaces results in the generation of an electrochemical gradient and
H+ flows down its concentration gradient to areas of low H+ concentration in the mitochondrial matrix
The flow of H+ through complex V or ATP synthase, releases energy which is captured by ATP synthase which generates ATP from ADP and P. This final step is oxidative phosphorylation.
At complex 4, oxygen accepts the electrons and is reduced to H20 (with the addition of 2H+), it is the final electron acceptor and why we need to breathe
respiratory poisons such as cyanide compete with oxygen?

Question 2

How do mitochondria appear in the cell?

Answer 2

Mitochondria exist as dynamic reticular networks, moving around by microtubules

Question 3

Complex 1

Answer 3

Is the largest complex, made up of 45-47 structural components , nearly a mega-dalton in size

Question 4

Complex 2

Answer 4

Small only 4 subunits

Question 5

Complex 3

Answer 5

11 subunits

Question 6

Complex 4

Answer 6

13 subunits

Question 7

Complex 5

Answer 7

16 subunits

Question 8

Vast majority of the proteins that make up these complexes are encoded on which genome? Why is this important?

Answer 8

Chromosomal DNA, only 13 of these polypeptides are encoded by mitochondrial genome.
It’s important because you need both chromosomal and mitochondrial DNA for synthesis of the subunits for proper function of the etc and oxidative phosphorylation

Question 9

In humans/mammals what is mtDNA like? Shape, size, copy number, inheritance,

Answer 9

Small molecule 16.6kb
First genome sequenced by Fred Sanger?
It is a covalently closed molecule (2 strands)
Highly organised structurally - it doesn’t have have introns, everything codes for something - therefore if so,e removed there’s consequences
We have 100-1000 mtDNA, tissues which required lots of energy such as muscle have higher copy numbers
Fibroblasts which are glytolytic have lower copy numbers
It is exclusively maternally inherited
It is vulnerable to free radical damage by ROS which makes it vulnerable to mutations

Question 10

Why is mtDNA vulnerable to free radical damage?

Answer 10

It is located near the respiratory chain which produces lots of free radicals
And does not have histones which protect nuclear DNA
IT does have some proteins

Question 11

Where is mtDNA located? And why does this matter?

Answer 11

It’s located in the mitochondria but all 13 peptides are mediated by nuclear gene products, therefore there has to be cross talk.

Question 12

So what does mtDNA code for (how many genes, proteins, what?)

Answer 12

37 genes 
13 proteins 
Which are structural components of oxidative phosphorylation and ETC
Complex 1 - 7 ND
Complex 4 - 3COX
Complex 3 - CYT b
Complex 5 - ATPase 6 and 8
Also encodes 2 rRNAs required for tln - 
22tRNAs required for tln and synthesis of proteins?

Question 13

mtDNA how does it repilicate?

Answer 13

No introns but tRNA punctuates protein coding regions
Small part of genome doesn’t code:
1. D-loop which contains ORI subscript H which has binding sites for txn factors, RNA pol, all nuclear encoded factors. Doesn’t encode a gene but important!
2. ORI ss L

Question 14

Why are we talking about mt? How many proteins are in it?

Answer 14

~1300 (13 from mtDNA, rest nuclear imported)

Mutations in the genes encoding these proteins cause human disease

Question 15

Mt disease is broad, can cause neurological disease and non-neurological? Name five neurological ones

Answer 15

Retinitis pigmentosa
Seizures
deafness
Peripheral neuropathy
Myopathy

Question 16

Mt disease is broad, can cause neurological disease and non-neurological? Name six non-neurological ones

Answer 16

Respiratory failure
Cardiomyopathy
Liver failure
Marrow failure
Diabetes
Thyroid disease

Question 17

Mt mutations can… (Finish the sentence)

Answer 17

Give rise to any symptom, in any organ or tissue (maybe one, maybe many), with an mode of inheritance (AR, X, AD)

Question 18

Nuclear-mt interactions

Answer 18

Mt has to be replicated and genes have to be txn/tln, lots of proteins are involved in this.

Question 19

There are more nuclear encoded mt proteins, therefore more defects in nuclear. However we to focus on mt defects. How are these different?

Answer 19

Milder and affects adults

Nuclear are more often recessive, severe and affect children.

Question 20

What kinds of mtDNA mutations are there?

Answer 20

Mutations in protein synthesising genes (tRNA, rRNA)

Mutations in protein coding genes (multisystemic NARP/MILS, tissue specific LHON

Question 21

What kinds of nuclear DNA defects are there?

Answer 21

Mutations in respiratory chain subunits, defects of mt protein synthesis, defects in lipid metabolism defects in apoptosis

Question 22

5 reasons why mt is at risk to mutate and cause disease?

Answer 22

High mutation rate (10-20x more than nuclear DNA)
No protective histones
Poor repair mechanisms
Vulnerable to ROS attack
No introns/little redundancy. Mut will affect a coding sequence

Question 23

We see a range of mutations occurring at any site round 16.6kb genome. Different genotype a but similar phenotypes. E.g.

Answer 23

Deafness, MELAS, LS

Question 24

Why are there common mutations? And why do they affect similar cells/tissues) MHTMs

Answer 24

The genetic rules of mtDNA!
Maternal inheritance
Heteroplasmy
Threshold effect
Mitosis segregation

Question 25

What is maternal inheritance? Why is it important?

Answer 25

MtDNA is only inherited from mum
2 reasons for this:
1. Paternal mt is diluted
2. Paternal mt is degraded
Important because - most mt diseases are maternally inherited, also as uni-inherited useful for looking at evolution

Question 26

Heteroplasmy? Importance?

Answer 26

In mt there are many copies of mtDNA
In non disease these are are the same, homoplasmic
In disease there are two populations, heteroplasmic (wt and disease)
Important because mt mutations are functionally recessive, meaning you need high no. Of mutant mtDNA to show disease

Question 27

Threshold effect

Answer 27

Heteroplasmic mutations are mainly recessive
High levels of mutation can be tolerated until it causes defect
Important in clinical phenotype expression
Look at CYT c activity and level of mutation, no. Mutations can increase and no change in CYT c function until threshold is met and then there is a quick drop in activity.

Question 28

Mitotic segregation?

Answer 28

Levels of mtDNA can vary enormously between tissue and individuals. At mitosis, we and mutant segregate randomly, affecting disease expression and inheritance. E.g. A mothers oocyte with high level of mutation can mature into a primary oocyte and by mitochondrial genetic bottle neck, results in an oocyte with with high, med. low or no mutation. Prediction of inheritance is hard!