Module 4.4

Question 1

Explain the theory of endosymbiosis and how this event gave rise to mitochondria.

Answer 1

• Describes how a large host cell (eukaryote) and ingested bacteria (prokaryote) became dependent on each other for survival, resulting in a symbiotic relationship
• The nucleus may have formed from invaginations of the plasma membrane around the nucleoid of an ancient prokaryote

Question 2

Describe the features of modern-day mitochondria that have been inherited from their prokaryotic ancestor.

Answer 2

Modern-day mitochondria arose from an oxygen-breathing bacterium

Question 3

Explain the steps in the life of a nuclear-encoded mitochondrial precursor.

Answer 3

• The 13 proteins that are maintained by the mitochondrial DNA are all components of the mitochondrial electron transport chain (ETC)
• This is due to their extreme hydrophobicity which would put too much pressure on the eukaryotic cell to produce them in the cytosol then transport them into the mitochondria from the outside
• A nuclear encoded mitochondrial precursor is a protein that is synthesised in the cytosol and then imported into the mitochondria
• The life of a nuclear-encoded mitochondria precursor
• Step 1: Nuclear encoded proteins are synthesised as “precursor” proteins on cytosolic ribosomes (precursor protein: an inactive protein form that can turn into an active form by post-transational modification – a building block for other proteins)
• Step 2: precursor proteins have targeting sequences or “zip codes” that deliver them to mitochondria
• Step 3: at mitochondria, precursor proteins engage with machines called “translocases” which mediate their entry into the organelle
• Mitochondrial Translocases
• TOM: Translocase of the Outer Membrane
• TIM: Translocase of the Inner Membrane
• SAM: Sorting and Assembly Machinery of the Outer membrane
• OXA1: Oxidase Assembly Protein 1
• Protein Transport in Cells:
• Targeting to each mitochondrial compartment utilises specific targeting signals
• Modes of transport for entry into mitochondria is transmembrane based (transmembrane: a type of integral membrane protein that spans the entirety of the cell membrane)
• The mitochondrial proteome:
• Proteome refers to the entire set of proteins that is, or can be, expressed by a genome, cell, tissue, or organism at a certain time
• Mitochondrial proteome refers to the entire set of proteins that is, or can be, expressed by the genome (including mitochondrial genome) and is targeted to mitochondria

Question 4

Recognize the clinical features of mitochondrial disease and describe the difference between primary and secondary mitochondrial disease.

Answer 4

• Mitochondrial disease (Mito)
• Long-term, genetic, often inherited disorders that occur when mitochondria fail to produce enough energy for the body to function properly
• Disease of energy production
   Clinically and genetically heterogenous meaning it can be caused by different genetic mutations
   Any symptom, any organ, any age, any mode of inheritance
• Mutations leading to Mito can be:
   Inherited from parents
   Can happen for the first time in a child (de novo mutations)
• Clinical Features:
   Mito principally affect tissues that are heavily reliant on oxidative metabolism
• The central nervous system
• Peripheral nerves, eye
• Skeletal and cardiac muscle
• Endocrine organs
   Many individuals with Mito have a multi-system disorder that often involves skeletal muscle and the central nervous system, but some individuals have a disorder that only affects one organ system
• Primary vs Secondary Mitochondrial Disease
   Primary mitochondrial disease (PMD) – genes with a primary link to the function of the electron transport chain (including mtDNA mutations)
   Secondary mitochondrial disease (SMD) – genes with an indirect function on the electron transport chain, or linked to another mitochondrial function

Question 5

Describe the difference between biochemical and genetic diagnosis approaches of mitochondrial disease.

Answer 5

• Biochemical Tests: Blood and urine studies are often the first step in diagnosing mitochondrial disease
• Include measurements of lactate and pyruvate in plasma, cerebrospinal fluid (CSF), and urine, as well as measuring specific amino acids and organic acids
• Other tests may be included: neuroimaging, electromyography (EMG) to measure muscle activity, and nerve conduction studies (NCS)
• Genetic studies of mitochondrial and nuclear DNA have replaced muscle biopsies as the gold standard for diagnosis
• Genetic testing is expensive and requires a good deal of evidence that the cause of symptoms is mitochondrial before insurance will cover this level of testing
• Things to consider:
• Clinicians are using muscle and tissue biopsies less frequently for mitochondrial diagnosis as they may not be as comprehensive as genetic testing and well tolerated by patients
• Also, PMD and SMD can’t be differentiated with tissue testing alone, functional tests – evaluations of how mitochondria are functioning in cells remain an important measure of mitochondrial function

Question 6

Define OMICS approach

Answer 6

• Functional studies in biomedical research:
• OMICS refers to the analysis of large amounts of data representing an entire set of some kind, especially the entire set of molecules, such as proteins, lipids, or metabolites, in a cell, organ, or organism

Question 7

Explain homoplasmy and heteroplasmy and the relevance to mitochondrial disease.

Answer 7

• Maternal inheritance of mtDNA
• Homoplasmy refers to the state of having uniformly normal or abmormal mitochondria in a tissue
• Heteroplasmy refers to the state of having a mixture of normal and abnormal mitochondria in a tissue
• The mitochondrial genome (mtDNA)
• Double-stranded, supercoiled circular molecule
• 37 genes in human cells - 2 rRNA, 22tRNA and 13 proteins
• Arranged in “nucleoids” mtDNA and proteins involved in replication and transcription
   Nucleoid: mammalian mtDNA is packaged in DNA-protein complexes denoted mitochondrial nucleoids
   Heteroplasmy: the presence of more than one mtDNA type in an individual

Question 8

Identify pathway names (not functions) affected in primary or secondary mitochondrial disease.

Answer 8

PMD:
- ETC (oxidative phosphorylation)
SMD:
- cholesterol and lipid metabolism pathways
- Calcium signalling pathways

Question 9

Describe the clinical features and understand the biological basis of Sengers syndrome - caused by nuclear DNA mutation

Answer 9

Nuclear gene mutation and mitochondrial disease:

• Case study: AGK, Sengers Syndrome and the power of Fundamental Biomedical Research
• Developing a protocol to understand the proteins that make up the human TIM22
   Cell culture line expressing hTim10bTAG
   Isolate (purify) TIM22 from cells using a technique called immune-precipitation
   Identify subunits using mass spectrometry
• Techniques:
   Cell culture: growth of cells from an animal or plant in an artificial environment, provides an excellent model system to study physiology or biochemical processes
   Immunoprecipitation: the technique of precipitating a protein antigen out of solution using an antibody (immune) that specifically binds to that particular antigen. Used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins
   Mass spectrometry: an analytical technique that accurately measures the mass of different molecules within a sample. In biological research, it can help us identify proteins within a sample
• The Acylglycerol Kinase (AGK)
   Lipid kinase (phosphorylate lipids in the cell, both on the plasma membrane as well as on the membranes of the organelles)
   Mutations in AGK cause a rare mitochondrial disease, Sengers syndrome
   Autosomal recessive
   Congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis
• Ultimately led to the knowledge that Senger Syndrome is also due to perturbations in protein import and not just lipid biogenesis

Question 10

Describe MELAS - an mtDNA mutation example cause of mito

Answer 10

• MELAS – Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes – A type of mitochondrial disease
• A rare genetic condition that begins in childhood
• The disorder affect many areas of the body, especially the brain and nervous system
• Early symptoms are seizures, recurrent headaches, loss of appetite and recurrent vomiting
• Stroke-like episodes with temporary muscle weakness on one side of the body (hemiparesis) may also occur leading to altered consciousness, vision and hearing loss, loss of motor skills and intellectual disability
• Causes of MELAS:
   Mutations in the mtDNA gene MT-TL1 in 80% of cases
   MT-TL1 gene provides instructions for making a molecule called a transfer RNA (tRNA)
   Transfer RNAs help assemble protein building blocks (amino acids) into functioning proteins
   The MT-TL1 gene provides instructions for making a specific form of tRNA that is designated as tRNALeu(UUR)
   Mutations in a nuclear gene (POLG1) have been associated with MELAS syndrome in one case

Question 11

• Mitochondrial diseases can arise from mutations in two genetic locations:

Answer 11

• mitochondrial DNA (mtDNA)
• mutations within nuclear genes that encode mitochondrial proteins
• Mitochondrial diseases can arise from mutations in two genetic locations:
• mitochondrial DNA (mtDNA)
• mutations within nuclear genes that encode mitochondrial proteins
• Mitochondria can’t be created de novo and therefore they are divided in by mitochondrial fission
• Mitochondrial DNA (mtDNA):
• When a cell divides, mitochondria are partitioned between the two daughter cells
• Mitochondrial segregation occurs randomly and is much less organised than the highly accurate process of nuclear chromosome segregation during mitosis
• Daughter cells receive similar, but not identical, copies of their mitochondrial DNA
• One cell contains numerous mitochondria, and each mitochondrion contains dozens of copies of the mitochondrial genome
• Human mtDNA disease is strictly maternally inherited
• mtDNA has a higher mutation rate than the nuclear genome, this leads to a heterogeneous population of mtDNA within the same cell and even within the same mitochondrion, as a result, mitochondria are considered heteroplasmic