Presentation on theme: "The Biochemistry of Adrenoleukodystrophy (ALD)"— Presentation transcript:
1 The Biochemistry of Adrenoleukodystrophy (ALD) Paul Watkins MD, PhDKennedy Krieger Institute &Johns Hopkins UniversityBaltimore, MD
2 Before we begin…ALD is a very complex disease, both clinically and biochemicallyWe have known about the basic biochemical defect since the 1980sThe ALD gene was discovered in 1993but…Many questions remain unanswered
4 Some basics… What is Biochemistry? The chemistry of biological systems The chemistry of lifeNeed to know a little bit of biology...Need to know a little bit of chemistry...
5 Chemical Elements Carbon (C) Hydrogen (H) Oxygen (O) Nitrogen (N) Living things are composed of several chemical building blocks, called “elements”The most abundant elements are:Carbon (C)Hydrogen (H)Oxygen (O)Other important elements include:Nitrogen (N)Sulfur (S)Phosphorus (P)
6 Chemical Bonds Elements can link together by forming chemical “bonds” Substances containing elements linked together by chemical bonds are called “Compounds”There are rules that must be followed:Carbon (C) can bond to 4 other elementsHydrogen (H) can only make 1 bondOxygen (O) bonds to 2 other elementsNitrogen (N) bonds to 3 other elements
7 Chemical compounds: some examples Water (H2O) H-O-HAmmonia (NH3)H-N-HHOxygen (O2) O=OIn oxygen, there are 2 lines betweenthe Os. This is a stronger “double bond”Methane(natural gas)(CH4)H-C-HH
8 Biochemical Problem in ALD Too much Very Long Chain Fatty Acid (VLCFA)Buildup of VLCFA is in all parts of the body, especially:Brain & spinal cordAdrenal GlandsVLCFA’s in blood plasma allows diagnosis of ALD by blood test
9 So what is a Fatty Acid? A Fatty acid is also a chemical compound Made up of 3 chemical elementsCarbon (C)Hydrogen (H)Oxygen (O)
10 Fatty Acids “Chains” of C and H form the “fatty” part of a fatty acid: Fatty Acids can have a chain that is...Short: 2-4 C’sMedium: 6-10 C’sLong: C’sVery Long: 22-? C’s
11 Fatty Acids C, H, and O bond together to form the “acid” part of a fatty acid:O-C-O-HNotice that while C is bonded to the O onthe right by a single line (bond), it isbonded to the O above by 2 lines (a“double bond”).
13 What does the body do with Fatty Acids? Like carbohydrates (sugars), fatty acids are used as a “fuel” for the body:Breakdown of fatty acids Energy + Heat + C, H, and O that are recycledThe energy released from FA breakdown is stored as ATP, which is used to drive muscle contraction, heart pumping, breathing, brain function, and many other processes
14 Popular misconception: Fatty acid molecules (including VLCFA) are freely floating around in our bloodstream, cells, and organsReality:99% of fatty acids are found in the more complex molecules shown in the next slide
15 What does the body do with Fatty Acids? Fatty acids are used to make complex fats (“lipids”), and fatty acids are the “building blocks”Some examples of complex lipids:Triglycerides – the fat on a piece of meat (or on us!)Phospholipids – the molecules that make up the membranes that surround all our cells and tissuesGlycolipids & Sphingolipids – molecules found in brain cells, especially in myelin membranes, that are necessary for normal brain functions
16 VLCFAsLike other fatty acids, VLCFAs are normal components of the body and are necessary for life.VLCFAs – important part of brain cell membrane lipids, especially myelin
17 VLCFAs – where do they come from? VLCFAs come from:DietElongation of shorter fatty acids(= “synthesis”)
18 VLCFAs (contd.)The balance between “just enough” and “too little” or “too much”Intake +synthesisExcretion +breakdowntoolittletoomuch
19 VLCFAs (contd.) Likely causes of excess VLCFA in ALD: Body makes too muchBody cannot remove excessEarly studies showed that the process whichdegrades VLCFAs (“b-oxidation”) was defectivein cells from ALD patientsIntake +synthesisExcretion +breakdowntoomuchlittle
20 * * * * * * * Fatty acid break- down (b-oxidation): removal of 2 carbons at a timeO-HO-H***O-HO-HO-H**O-HO-H*Four chemical reactions**O-H
21 VLCFAs (contd.) More recent studies indicate that VLCFA synthesis (elongation of long-chain FAs, orLCFA) is also increased in cells from ALDpatientsIntake +SYNTHESISExcretion +breakdowntoomuchlittle
22 VLCFA Synthesis Dietary LCFAs O 16 C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-O C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OO161820222426DietaryLCFAs
23 FA activationTo do either process – break down VLCFA or make them from LCFA – the FAs first need to be “activated”Activation means attaching the FA to a helper molecule called “Coenzyme A” (or CoA for short)We would write this as “LCFA-CoA” or “VLCFA-CoA”(Remember this when we get to Part 2!)
24 A brief summary…Fatty acids are chemical compounds made of C, H, and OVLCFA are ~50% longer than LCFA that are abundant in our normal dietThe elevated VLCFA levels in ALD come from both increased synthesis and decreased degradationUnanswered questions…Which is more important?How does any of this relate to the ALD gene and protein?
29 The X-ChromosomeThe ALD gene is found at the tip of the longer arm (“q”) of the X-chromosomepq
30 DNA Chromosomes are mainly DNA The “Double helix” of DNA is made up of 4 chemicalscalled “bases”:AdenineCytosineGuanineThyminePairing of T on one strand with A on the other, or C with G, causes the double helix to formA single strand of DNA is millions of bases long
31 DNA: a blueprint for making proteins DNA is made up of the 4 bases: A, C, G, and TThe order, or “sequence”, of As, Cs, Gs, and Ts on astrand of DNA is really the “instructions” for making proteinsA GENE is a piece of DNA that has the instructions formaking one specific proteinProteins, like DNA, are long chains of chemicalsWhile DNA is a chain of bases, proteins are chains ofAMINO ACIDSThere are 20 different amino acids
32 From DNA to protein…Since DNA has only 4 bases, but there are 20 differentamino acids, how does the DNA code work?A group of 3 bases in the DNA sequence is called a“codon” that specifies one specific amino acidThere are also “start” and “stop” signals in the code“Messenger RNA” or mRNA serves as an intermediate inbetween DNA and protein
33 mRNA DNA mRNA mRNA has a single strand It faithfully copies the base sequence of one of the DNA strandsIn RNA, the base Uracil is used instead of Thymine
34 Folded protein molecule From mRNA to protein…mRNACodonsAmino acidsFolded protein moleculeThe ALD protein has 745amino acids!How many bases are neededto encode this protein?
35 The ALD gene Known to be near the tip of the long arm of the X-chromosome“Discovered” in 1993 by Drs. Aubourg, Mandeland coworkers:Location pinpointed more accuratelySequence of As, Cs, Gs, and Ts (bases)determinedThe “instructions” for making the ALDprotein require 2238* bases!*For math geeks who know that 745x3=2235 and not 2238,we have to add 3 more bases for the stop signal
36 The ALD gene All ALD patients have a “mutation” in their ALD gene: An incorrect nucleotide (for example, a “T”where an “A” should be)One or more nucleotides missingOne or more extra nucleotides
37 The ALD proteinWhen there is a mutation in the ALD gene, what happens to the ALD protein?Several possibilities:A single incorrect amino acid gets placed in the protein (may or may not affect function)The amino acids are correct up to a point, but then after that they are all incorrect (definitely affects function)The amino acids are correct up to a point, but then we run into a premature “stop” signal (definitely affects function)
38 The ALD protein The ALD protein contains 745 amino acids Its amino acid sequence was determined from the sequence of bases in the ALD geneALD protein amino acid sequence was compared to that of other known proteinsThe protein defective in ALD was not any of the enzymes known to participate in the breakdown or synthesis of VLCFAThe ALD protein belongs to a large family of proteins called “ABC transporters”
39 Getting something from one ABCtransporters:Getting something from oneside of a membrane…membranetransporter
41 Where is the ALD protein found inside cells? The ALD protein is located in the membrane surrounding “peroxisomes”What are peroxisomes?
42 Cells and their internal structures Smaller compartments within cells are called “organelles”Some of these are:EndoplasmicreticulumMitochondriaCellmembraneNucleusPeroxisomesLysosomes
43 Cells and their internal structures Biochemical processes take place within specific organellesDegradation (breakdown) of VLCFA occurs in peroxisomesSynthesis of VLCFA via elongation of LCFA occurs in the endoplasmic reticulumOften, the internal location of a biochemical process is important for understanding how disease results
44 The ALD protein: What does it do? VLCFA-CoARecent work from the labs of Ronald Wanders and Stefan Kemp in Amsterdam show that the ALD protein transports the “activated” form of VLCFA (VLCFA-CoA) into peroxisomes, where VLCFA breakdown occurs.cytoplasmperoxisomeVLCFA-CoA
45 Putting it all together – Normal cells VLCFA synthesis(elongation)ALDProteinBreakdown of excess
46 are used as building blocks Putting it all together – ALD cellsVLCFA accumulate andare used as building blocksto make complex lipidsXVLCFA synthesis(elongation)ALDProtein
47 Another brief summary… Chromosomes are structures in the nucleus of cells that are made up of thousands of genesA gene is a piece of DNA containing “instructions” for making one specific proteinIf the gene has a mistake in its DNA, the protein that it codes for will often be defectiveThe ALD gene encodes the ALD Protein (ALDP)ALDP is now thought to transport VLCFA-CoA into peroxisomes where degradation takes place
51 Prevention - Newborn screening “Blood spots” on thick paper cards are obtained from all newborn infantsAll states screen for phenylketonuria (PKU) and other treatable genetic diseasesRecent results from the Moser lab demonstrated that screening for ALD is feasible?Coming soon to a state near you?
52 Treatment – Diet (Lorenzo’s oil) Some definitions:“Saturated” fatty acid (no double bonds):-C-C-C-C-C-C-C-Saturated VLCFA are toxic to cells“Mono-unsaturated” fatty acid (one double bond):-C-C-C-C=C-C-C-C-Monounsaturated VLCFA are far less toxic“Poly-unsaturated” fatty acid (more than one double bond: -C-C-C=C-C-C=C-C-CAlso less toxic
53 Treatment – Diet (Lorenzo’s oil) Lorenzo’s oil is a mixture of triglycerides containing monounsaturated fatty acidsBecause monounsaturated fatty acids are there in excess, the body preferentially uses them to synthesize less-toxic monounsaturated VLCFA (rather than toxic saturated VLCFA) by the elongation process shown in slide #22Patients taking Lorenzo’s oil have reduced plasma levels of saturated VLCFA, but high levels of mono- unsaturated VLCFA
54 Bone Marrow Transplantation Rationale for BMT:Give patient someone else’s cells which contain good enzymeProcedure:Find donorDestroy patient’s bone marrow cellsGive patient bone marrow from donor
55 Bone Marrow Transplantation Potential problems:Finding a donorRisky – MortalityRejectionGraft vs. host diseaseIn which patients is BMT more likely to work?Fairly strict selection criteria
56 Treatment – Gene Therapy Hope for the present and futureProblems:Get gene to correct cells and keep it thereOnce there, get it to work properlyMake sure there are no harmful side effectsRemember, too much of a good thing can be just as bad as not having enough…
57 The final summary…Knowing something about the biochemistry of ALD allows us to perform tests that are useful in the prevention of ALDKnowing something about ALD biochemistry has also facilitated development of therapies such as Lorenzo’s oil, bone marrow transplantation, and gene therapyKnowing something about the biochemistry of ALD also allows us to search for drugs that may, in the future, be beneficial for treatment