VAP-B's role in ALS and its P56S mutation
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VAP-B and its role in Amyotrophic lateral sclerosis The document discusses VAP-B and its role in Amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disorder characterized by motor neuron death. The P56S mutation in VAP-B has been linked to familial ALS and causes protein aggregation, ubiquitination, and insolubility. The mutation induces conformational changes in VAP-B that enhance oligomerization and prevent interaction with lipid transfer proteins, impairing membrane trafficking and lipid homeostasis. This leads to endoplasmic reticulum stress, proteasomal dysfunction, and motor neuron degeneration.
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VAP-B's role in ALS and its P56S mutation
- 1. VAP-B and its role in Amyotrophic lateral sclerosis
- 2. Amyotrophic lateral sclerosis (ALS) No cure or effective treatment Rapidly progressive neurodegenerative disorder characterized by death of motor neurons in the cerebral cortex, brain stem, and spinal cord. Two forms :Sporadic (~90%), Familial (10%)- 20% of familial cases are caused by dominantly inherited mutations in SOD1 Prevalence of 4-6 per 100,000 population, and an average mortality of 5 years after onset. Most studies on the familial form have been focused on SOD1 A complex interplay between multiple pathogenic processes including, Oxidative Stress, Protein Aggregation, Mitochondrial Dysfunction excitotoxicity, Impaired Axonal Transport
- 3. 1251 5942 211158 243220 VAPB-1 H. sapiens LSTR-LLALVVLFFIVGVIIGKIAL aVAP33 A.californica VRAFPPVVYVVAAIILGLIIGKFLL DVAP33A D. melanogaster QIPVFYIAVAIAAAIVSLLLGKFFL VAP homolog C.elegans YPTLQIFLIAVAALLIGLIVGRLF- VAP27-1c A.thaliana IPFMYVLLVGLIGLILGYIMKRT-- SCS2 S.cerevisiae SSMGIFILVALLILVLGWFYR---- VAP homolog D.discoideum FNHYQTLIVIFAIAILSFIFGKLI- CCDMSP Domain VAP consensus sequence VAPB-1 H. sapiens FKVKTTAP--YCVRPNSG aVAP33 A. californica FKVKTTAP--YCVRPNSG DVAP33A D. melanogaster FKIKTTAP--YCVRPNIG VAP-homolog C. elegans FKVKTTAP--YCVRPNSG VAP27-1c A. thaliana FKVKTTNP--YCVRPNTG SCS2 S. cerevisiae FKVKTTAP--YCVRPNAA VAP-homolog D. discoideum FKVKTTAP--YCVRPNTG MSP78 C. elegans YGIKTTNM--LGVDPPCG VAPs-MSP homology * **** * * * TMD The P56S mutation in VAP-B causes motor neuron degeneration diseases GXXXG P56S The P56S mutation induces cis to trans isomerization
- 4. The underlying mechanism of VAP-B(P56S)-induced ALSThe underlying mechanism of VAP-B(P56S)-induced ALS The P56S mutation induces the formation of insoluble protein aggregates Triton X-100 RIPA β-tubulin VAP-B S P P56S S P S P S P S P S P S P P56S P56SWTWT- - IP: VAP-B P56S W T P56S W T Blot: anti-Myc anti-Myc anti-Myc FK2 IgG(LC) IgG(HC) The VAP-B(P56S) is polyubiquitinated
- 5. Wild-type VAP-B is recruited into VAP-B(P56S) aggregates S SP P HA-VAP-B(WT) Myc-VAP-B(P56S) VAP-B: P56SWT Potential mechanisms underlying the diseasePotential mechanisms underlying the disease The disease is inherited in an autosomal dominant fashion Loss of function Gain of toxic of function WT WTWTP56SP56SP56S P56SP56SP56SP56S UPR associated activity Misfolded conformation P56SP56SP56SP56S ub ub ub Proteasomal dysfunction ERAD ER Stress Motor Neuron Degeneration Motor Neuron Degeneration P56SP56SWT WT WTP56S Impaired lipid homeostasis & membrane trafficking FFAT
- 6. Structural requirements for VAP-B oligomerization and their contribution for VAP-B(P56S) aggregation and neurotoxicity Structural requirements for VAP-B oligomerization and their contribution for VAP-B(P56S) aggregation and neurotoxicity VAP-B (WT)MSP CC GXXXG mutant (II)MSP CC 169 112 92 67 59 36.5 31 Monomer Dimer Tetramer VAP-B(WT)VAP-B(I)VAP-B(II)K87/M89D K87/M89D/(I)K87/M89D/(II) YFP-TMD(II) YFP-TMDYFP YFP Monomer Dimer 169 112 92 67 59 36.5 31 YFP-TMD: WT II WT II CL CL-Rev. The GXXXG motif
- 7. VAP-B-∆MSPCC CC VAP-B-∆MSP (II) ∆MSP-Myc: WT II WT II CL CL-Rev. Dimer Monomer MSP Dimer Monomer MSP-Myc: CL WT DD P56S CL-Rev. WT DD P56S 59 36.5 31 VAP-B-∆CCMSP VAP-B-∆CC (II)MSP The Coiled-coil domain Monomer Monomer* Dimer CL Dimer* CL-Rev. ∆CC/II ∆CC II W T ∆CC/II ∆CC II W T P56S/∆CC ∆CC P56S W T P56S/∆CC/II ∆CC/II II P56S/II Monomer* Monomer Dimer Dimer* The MSP domain
- 8. The P56S induces conformational changes of the MSP domain Evidence: Mutation induces a ~18% increase in the average diameter size of the domain Suggests: Decrease in overall compactness Evidence: The mutant rests essentially in an agglomerate conformation at physiologic temperature!! Suggests: Patologic propensity to aggregate Evidence: Mutation induces secondary Structure alterations Suggests: Folding modifications
- 9. Evidence: Mutation induces exposure of hydrophobic patches Suggests: Hydrophobic core destabilization Evidence: ANS binding is decreased in VAP-P56S after incubation at 37ºC Suggests: P56S mutants can clump together, likely involving hydrophobic interactions, thus resulting in less available binding sites for ANS. This may indicate that hydrophobic interactions are involved in the formation of the agglomerates.
- 10. +VAP-B(P56S)-HA WT S P S P S P S P ∆CC∆CC/IIIIVAP-B-Myc: IB: anti-HA IB: anti-Myc IB: anti-Myc VAP-B S P S P S P VAP-B: WT P56S Nir2 CERT M SP(P56S) M SP(W T) P56S/∆CC/II M ock M SP(87/89D) +Nir2-HA IB:anti-HA IB:anti-HA IB:anti-Myc Lysate IP:anti-Myc The P56S mutation per se has no effect on FFAT binding. Rather, it enhances VAP-B(P56S) oligomerization thereby preventing FFAT binding. The P56S mutation per se has no effect on FFAT binding. Rather, it enhances VAP-B(P56S) oligomerization thereby preventing FFAT binding. The effect of the P56S mutation on FFAT binding
- 13. FFAT FFATFFAT MSP Formation of insoluble protein aggregates by the P56S mutation P56S P56SP56S P56SP56S P56SP56S MSP MSP P56SP56S P56SP56S Insoluble Aggregates
- 14. Amir Kedan M.Sc. student Omer Keinan Ph.D. student Orly Laufman Ph.D. student Acknowledgment Koret Hischberg -Tel-Aviv University Zvulun Elazar-Weizmann Institute Tony Futerman-Weizmann Institute Moti Liscovitch -Weizmann Institute Chaya Brodie- Bar-Ilan University Sean Munro-MRC, Cambridge, UK Kentaro Hanada-Tokyo, Japan Antonella De Matteis- Mario Negri, Italy Michael Selitrennik Research associate So Hui Kim Ph.D. student Dr. Diego Peretti Postdoctoral fellow Dr. Cláudio M. Gomes Dr. Sónia S. Leal Collaboration Tecnologia Química e Biológica (ITQB), University Nova, Lisboa, Portugal Former Roy Amarilio Sreekumar Ramachandran Vladimir Litvak Nili Dahan Donghua Tian
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