EAST 501 Lecture Notes - Lecture 13: Monocarboxylate Transporter 8, African Clawed Frog, Aromatic Amino Acids
21 Jun 2018
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L13 P4 03/26
Monocarboxylate Transporter 8 and Allan-Herndon Dudley Syndrome
1
OVERVIEW
- Introduction
o Thyroid, TH, TH Transporters, MCT8
- MCT8 throughout the body
- Pathology
o AHDS characterization
o MCT8 KO models and CNS development
- Treatment
o Thyroid Hormones Based and Novel Putative Therapy
THYROID AND THYROID HORMONES
- Thyroid Hormones (TH) required for normal brain and somatic tissue development in the fetus and newborns
and regulates protein, carbohydrate, and fat metabolism in everyone (all ages)
- The main hormones are thyroxine (T4) and triiodothyronine (T3)
- Believed for a long time that TH crosses plasma membrane by passive diffusion
- T-type amino acid transporter (TAT1) was cloned
o Transports aromatic amino acids but not iodothyronine (involved in conversion from T4 to T3)
o TAT1 was found to be a member of the monocarboxylate transporter (MCT) family (MCT10 and MCT8)
and is most homologous with MCT8 (then they started evaluating MCT8 as a potential thyroid hormone
transporter)
MCT8 TH TRANSPORT ACTIVITY
- Coding sequence of rat MCT8 was cloned using rtPCR from rat liver and injected into xenopus laevis oocytes
(African frog)
- MCT8 expression at the plasma membrane
o Immunofluorescence microscopy was performed on oocytes à strongly expressed
- Transport
o Oocytes injected with MCT8 cRNA showed increase in uptake of T3 and T4 (showed that it allows T3 and
T4 to be transported in)
o Substrate specificity
§ Incubated the oocytes with different radioactive ligands (T4, T3, rT3, and amino acids Leu, Phe
Tyr and Trp)
• rT4 – different iodine in T4 is cleaved off = different conformation of T4
§ MCT8 is specific to the T4 and T3
OTHER TH TRANSPORTERS
- Found later that several transporters from different protein families were capable of transporting
iodothyronines
o Organic anion transporting polypeptide (OATP)
o Na-taurocholate co-transporter (LAT1 and LAT2)
o Monocarboxylate Transporter (MCT) (MCT8 and MCT10)
MONOCARBOXYLATE TRANSPORTER 8 (MCT8)
- Major function of MCT8 is the cellular import and export of T4 and T3 along their concentration gradients across
the plasma membrane
o important for transport of T3 across the blood-brain barrier
- Located on Xq13/2 chromosome
- mutations in MCT8 are responsible for a rare X-linked disorder, Allan-Herndon-Dudley syndrome (AHDS)
L13 P4 03/26
Monocarboxylate Transporter 8 and Allan-Herndon Dudley Syndrome
2
LONG VS. SHORT MCT8
- 6 exons coding for protein with 12 TM domains
- Unique to MCT8 is presence of PEST(aa code) domains in N-terminal à create long MCT8
o there are two translational start sites à one for long and one for short MCT8 to be created
- Significance of long vs. short
o don’t really know the significance of long vs. short MCT8
o patient with psychomotor retardation had mutation in the translation site that allowed for long MCT8,
but also found healthy person with same mutation – the mutation was the same, but one was healthy
and one not
- Recent study suggested if long MCT8 is generated, it undergoes effective ubiquitination and proteasomal
degradation
o Maybe useful for turnover of the MCT8 protein
- Most species only express short MCT8 due to lack of one of the two translational start sites
- Functional studies of human MCT8 have been carried out using short MCT8
T3 DOCKING SITE FOR MCT8
- 3 MCT8 models were created: extracellular open, extracellular partly occluded and intracellular open
o conformations were used for T3-docking studies
- One central substrate binding site
o Formed and shaped by transport sensitive residues His192, His415, Arg445, Asp498, and Cys497
o New models revealed that these residues are accessible both from the extracellular and intracellular
side in conformations open towards these compartments
T3 DOCKING SITE FOR MCT8 – MECHANISM
- After extracellular recognition, T3 adheres alternately to these residues at binding sites
- The N- and C-terminal domains are tilting around the binding site with alternating extra and intracellular
accessibility of the known transport sensitive MCT8 residues
- In doing so, the T3 binding sites likely undergo a conformational change and T3 is able to pass through
MCT8 EXPRESSION
- Human à determined using RT-PCR
o in brain: high in choroid plexus, capillaries and neurons
o Also, thyroid, liver and kidney
- Mouse
o brain, liver, kidney
§ liver – sinusoidal membrane and hepatocytes
§ kidney – basal membrane of proximal tubule
§ brain – neurons and endothelial cells of capillaries in the brain and choroid plexus
- Localization is important for understanding mechanism of phenotypes associated with mutations in MCT8 so
that you know where to look
- MCT8 expression alters with advancing age
o brain development of mice
o livers of old rats
o human placenta with advancing gestation
- Elevated MCT8 expression was found in placenta
o placenta with severe intrauterine growth restriction
o in femurs of hypothyroid mice
o liver and skeletal muscle of critically ill patients and rabbits
L13 P4 03/26
Monocarboxylate Transporter 8 and Allan-Herndon Dudley Syndrome
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TAKEAWAYS
- MCT8 is important for transporting T3 and T4
o specific to those two THs
- Expressed mostly in liver, kidney and brain
- have long and short form – function of the long form is unknown
- Important for transporting T3 across blood brain barrier
ALLAN-HERDON-DUDLEY SYNDROME (AHDS)
- Caused by X-lined recessive mutations in MCT8 à mostly found in males
- Characterized by mental retardation and peripheral thyrotoxicity
SYMPTOMS
- early (first few months)
o Hypotonia o Feeding difficulties
- later (months, year)
o Limb rigidity & spasticity
o low muscle mass and generalized
muscle weakness
o Poor head control
o lagging weight gain
o microcephaly
o profound cognitive impairment
o Lack of speech
PATHOGENESIS
- Features of both TH excess and deficiency
o Probably due to tightly regulated expression of TH transporters and diodonases
- Deficiency
o Mental and neuromotor impairment
- Excess
o Hypermetabolic state
§ Difficulty to maintain weight § Difficulty to maintain muscle
mass
DIAGNOSIS
- TH profile in serum:
o high T3
o low rT3
o low T4
o normal to slightly elevated TSH
- diagnosis confirmed by MCT8 gene sequencing
MCT8 DEFICIENCY CAUSES AHDS
- Range of mutations:
o indels
o missense mutations
o nonsense mutations
- Results in
o lower expression of MCT8
o impaired trafficking to PM
o reduced specificity for THs
o loss of function
STUDY: PRENATAL BRAIN DAMAGE FROM MUTATED MCT8 IN FETUS AND 11 YEAR OLD BOY WITH AHDS
- Analyzed 30th gestational week male fetus and 11 year old boy with similar MCT8 mutations
o Boy had TH profile typical of AHDS
- pathologist compared the two brains
- Results support notion that impaired supply to TH to the brain starts at the prenatal stage is the cause of brain
damage observed in an adult
o found reduced levels of THs in the brain of both fetus and boy
Document Summary
Pathology: ahds characterization, mct8 ko models and cns development. Treatment: thyroid hormones based and novel putative therapy. Thyroid hormones (th) required for normal brain and somatic tissue development in the fetus and newborns and regulates protein, carbohydrate, and fat metabolism in everyone (all ages) The main hormones are thyroxine (t4) and triiodothyronine (t3) Believed for a long time that th crosses plasma membrane by passive diffusion. Coding sequence of rat mct8 was cloned using rtpcr from rat liver and injected into xenopus laevis oocytes (african frog) Immunofluorescence microscopy was performed on oocytes strongly expressed. Transport: oocytes injected with mct8 crna showed increase in uptake of t3 and t4 (showed that it allows t3 and. T4 to be transported in: substrate specificity. Incubated the oocytes with different radioactive ligands (t4, t3, rt3, and amino acids leu, phe. Tyr and trp) rt4 different iodine in t4 is cleaved off = different conformation of t4. Mct8 is specific to the t4 and t3.
