M3: Molecular bio of disease

• DEFECTIVE MITOCHONDRIAL TARGETING

  • Mitochondrial targeting sequences have a high content of positively charged residues(Arg, R) and has a strong tendency to form an amphiphilic a-helix
  •  Point mutation in the targeting sequence of a pyruvate dehydrogenase (PDH)complex subunit
  •  Proline is a helix breaker
  •  Inefficient import reduces levels of pyruvate dehydrogenase in mitochondria
  •  Pyruvate accumulates -> lactate -> build-up of lactic acid in blood
  •  Inherited metabolic disorder congenital lactic acidosis
• DEFECTIVE ER TARGETING

  • ER targeting sequence: more than or equal to 8 hydrophobic amino acids, often N-terminal, sometimes cleaved
  •  Recognised by SRP, targeted to ER membrane and cause Sec61 translocator channelto open, initiating translocation
  •  Point mutation in insulin signal sequence causes diabetes
  •  Does not interact correctly with Sec61 and not efficiently translocated into the ER
  •  Less insulin made
  • Incorrectly localised to cytosol, forms toxic aggregates causing beta-cell death
• PROTEINS THAT ARE INCORRECTLY TARGETED...

  • Localise incorrectly to cytosol (default localisation)
  • Broken down (degraded) into amino acids by proteolysis
• CYTOSOLIC PROTEASOME

  • Large protease complex which degrades proteins by proteolysis
  •  Short-lived proteins and misfolded proteins degraded
  •  Proteins marked for proteolysis by attachment of a protein called ubiquitin
  •  Polyubiquitin chain recognised by the proteasome
• PROTEIN FOLDING AND MISFOLDING

  • Newly synthesised proteins must fold into correct 3D conformation
  •  Protein folding is error prone Mutations that change amino acid sequence can prevent correct folding
  •  Many disease-causing mutations cause proteins to misfold
• MISFOLDED PROTEINS

  •  Retained in the ER by quality control as misfolded proteins are potentially harmful
  •  Chaperones bind to misfolded proteins and stop them leaving the ER: Quality control
  •  Mutant protein does not reach site of function
  •  Lack of functional protein can cause disease
• CYSTIC FIBROSIS

  • Recessive genetic disorder, affects 1 in 3000 live births, often fatal by the age of 40
  •  Caused by mutations in a chloride channel called CFTR (cystic fibrosis transmembrane conductance regulator)
  •  CFTR expressed in epithelial cells in many organs (lung, liver, pancreas)
  •  Pumps Cl- ions out of cells, water follows by osmosis
  •  Keeps mucus on surface of epithelial cells hydrated
  •  Cilia can beat to remove bacteria and debris
  •  CF mucus becomes dehydrated and cilia cannot function
• CFTR MUTATIONS

  • Most common mutation is deletion of phenylalanine at position dF508
  •  ~90% of patients have at least one copy of the dF508 mutant gene
  •  dF508 CFTR cannot fold correctly
  •  Retained by ER quality control system
  •  Does not reach plasma membrane
  •  Misfolded CFTR that are recognised and retained by ER quality control must beremoved, then moved back into cytosol and degraded by the proteasome
  •  ER-associated degradation: ERAD
• THERAPIES FOR CF

  • Misfolded protein degraded, loss of function
  •  Express wild type (i.e. normal) CFTR through gene therapy/gene editing
  • dF508 CFTR does still function as a Cl- channel
  •  Raises the possibility of using drugs to enhance its foldingand allow it to escape from ER quality control / ERAD
  •  Pharmacological chaperones
  •  Correctors as they correct the folding defect
  •  Lumacaftor, tezacaftor
• DISEASES ASSOCIATED WITH ACCUMULATION OF MISFOLDED PROTEINS IN THE ER

  • Build-up of misfolded proteins in the ER lumen
  •  Triggers the unfolded protein response (UPR)
  •  UPR attempts to restore homeostasis – increases expression of chaperones andinhibits protein synthesis
  •  If homeostasis cannot be restored, the UPR can trigger programmed cell death,apoptosis
  •  Proteotoxicity: Gain of function
• CHYLOMICRON BIOSYNTHESIS

  • Occurs in the ER and Golgi
  •  PreChylomicrons assembled in ER, triglycerides and large proteins (fatty acids from dietary lipids)
  •  Packaged into transport vesicles (PCTV) for delivery to Golgi
  •  Mature into chylomicrons in Golgi
  •  Released by exocytosis and enters capillaries
• CHYLOMICRON RETENTION DISEASE

  • In chylomicron retention disease (CRD), prechylomicrons accumulate in the ER and are unable to reach the Golgi
  •  Apoproteins that initiate assembly of prechylomicrons are properly folded and preCMs are correctly assembled
  •  Not an ER folding disease
  •  The COPII coat responsible for cargo export from the ER fails to assemble correctly
  •  This leaves the newly assembled preCMs trapped inside the ER and unable to progress to the cis Golgi
• SAR1 GTPASE

  • Controls the formation of COPII vesicles
  •  Sar1p in the cytosol is in GDP-bound form ‘off’
  •  A regulatory protein on the ER membrane activates Sar1p ‘on’
  •  Sar1-GTP initiates assembly of COPII coat proteins on ER membrane
• SAR1 GENES

  • Humans have 2 different isoforms of Sar1 genes:

  1. Sar1a
  2. Sar1b

  • They are 90% identical in amino acid sequence but encoded by different genes on different chromosomes
• MUTATIONS IN SAR1b

  • Cause CRD as Sar1b promotes export of prechylomicrons
  • 20 mutations linked to CRD have been identified in Sar1b gene
  • Effects are no Sar1p is made, GTP binding site is defective
  • Loss of Sar1b function prevents prechylomicrons export from ER
• LOSS OF SAR1b

  • Selectively disrupts prechylomicrons export
  • Sar1a is still active and functions to mediate vesicular transport of most cargo
  • Sar1b required for transport of specific cargo including prechylomicrons
  • Prechylomicrons are large so require large transport vesicles
• SYMPTOMS AND TREATMENT OF LOSS OF SAR1B

  • The defect in ER export results in accumulation of preCMs in the ER and theappearance of lipid droplets in the cytoplasm of cells in the intestine
  •  Impaired absorption of fats, cholesterol, and fat-soluble vitamins
  •  Slow growth and weight gain, effects on GI and nervous systems
  •  Treatment involved a low fat diet to minimise accumulation of intracellular preMCs
• FAMILIAL HYPERCHOLESTEROLEMIA (FH)

  • Autosomal-dominant disease
  •  Leading cause of coronary heart disease
  •  Heterozygous FH incidence 1:200 – 1:500 (UK ~1:270)
  •  Caused by defects in cholesterol uptake

  1. Cholesterol accumulates in blood -> atherosclerosis
• TREATMENTS FOR FH

  • Inhibit cholesterol synthesis:

  1. Stimulates LDL receptor expression
  2. Effective for heterozygotes who have one copy of wild type (normal) gene
  3. Increased uptake of LDL
  4. Statins inhibit HMG-CoA reductase

  • Inhibit dietary cholesterol absorption

  1. EZETIMIBE acts in the intestine
• LYSOSOMAL STORAGE DISEASE

  • >50 inherited metabolic diseases typically linked to defects in acid hydrolases andtransporters
  •  Progressive accumulation of uncleaved/untransported substrates results in a widerange of pathologies
  •  Niemann-Pick Type C: 95% of cases caused by mutations in the membrane proteinNPC1
  •  Gaucher disease: caused by mutations in the lysosomal acid Beta-glucosidase
• NIEMANN-PICK TYPE C

  • NPC1 is a membrane protein – synthesised at the ER and sorted to lysosomes
  •  Transports the cholesterol released from LDLs into the cytosol
  •  Most common mutation in NPC1 causes the protein to misfold and be retained in theER i.e. it is an ER misfolding disease
  •  Inefficient transport of dietary cholesterol out of the lysosome results in the cell trying to make more of its own cholesterol
  •  Disrupts production and degradation of various lipids that perturb a range of cellular functions
• MOLECULAR BASIS OF GAUCHER DISEASE

  • Most common mutations cause misfolding of lysosomal acid b-glucosidase causingretention in the ER
  •  Another example of an ER misfolding disease
  •  Absences of acid b-glucosidase causes the accumulation of glucosylceramide and other glycolipids in the lysosomes causing a range of symptoms
  •  Cleavage requires lysosomal acid b-glucosidase
• MOLECULAR BASIS OF GAUCHER DISEASE cont.

  • Monocyte-macrophage system is affected the most because of high amount of GLC in the cell lysosome, causing accumulation of Gaucher cells
  •  Production of inflammatory cytokines that can cause enlargement of the spleen and liver, abnormalities of the lung, destruction of bone and progression of anemia, thrombocytopenia and leukopenia
• POTENTIAL TREATMENTS

  • Enzyme replacement therapy :

  1. Inject synthetic enzymes that are taken up from outside the cell by endocytosis
  2. M6P receptors in plasma membrane and reach the lysosome via endocytosis

  • Substrate reduction therapy:

  1. Reduce amount of glycosylceramide in lysosomes – don’t need so much lysosomal acid b-glucosidase
  2. Misglustat inhibits glucosylceramide synthesis, used to treat Gaucher disease

  • Pharmacological chaperones

  1. Drugs that promote correct folding can increase the amount of enzyme that escapes ER quality control and reaches the lysosome