Le malattie da triplette

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1 Le malattie da triplette
28 Aprile 2010 Paola Rusmini

2 Una porzione significante del genoma umano contiene semplici sequenze ripetute come i MICROSATELLITI. I Microsatelliti sono : -Sequenze ripetute in TANDEM di 1-6 nucleotidi. -Templato per mutazioni dinamiche trovate in diverse malattie denominate MALATTIE da TRIPLETTE (malattie neurologiche).

3 Le mutazioni dinamiche:
-sono cambiamenti nel materiale genetico che possono continuare a mutare nei tessuti (mitosi) e lungo le generazioni (meiosi,più frequenti) -possono avere effetti sulla struttura del DNA, la trascrizione, splicing trasporto e stabilità dell’RNA, sulla traduzione, sulla struttura e funzionalità proteica

4 Pearson CE et al. doi: 10.1038/nrg1689
Unstable repeat tracts and the process associated with repeat instability Nature Reviews Genetics (2005) 6,

5 Malattie da triplette Espansione in regioni non codificanti (CGG;GCC,GAA,CTG,CAG) La mutazione causa perdita di funzione o danni a livello di mRNA. Espansione in regioni codificanti: La mutazione porta ad una proteina mutata con guadagno di funzione tossica e/o perdita di funzionalità.

6 La lunghezza del tratto espanso:
Dipende dai processi metabolici del DNA. (duplicazione-Riparo e mantenimento perché è un fenomeno presente anche in cellule post-mitotiche). Da luogo al MOSAICISMO genetico Tratti più lunghi sono più soggetti a mutazioni di quelli corti ANTICIPAZIONE GENETICA

7 Gusella JF & MacDonald ME
Unmasking polyglutamine triggers in neurodegenerative diseases Nature Reviews Neuroscience 2000, 1:

8 Sequenze ricche in glutammina/aspartato (Q/N):
-Sono presenti frequentemente negli eucarioti. Si ritrovano nei lieviti e il loro numero aumenta negli invertebrati e nei mammiferi, indicando che gli organismi più complessi necessitano di queste sequenze ripetute. -Non sono più abbondanti nelle proteine neurologiche -Si ritrovano in proteine importanti per lo sviluppo, e in domini proteici funzionali (interazioni proteiche-riconoscimenti molecolari-signalling). -Non si conosce ancora il loro significato, vi sono diverse ipotesi: Per modulare nuovi domini proteici Per modulare lo sviluppo e aggiungere nuove diversità nella specie.

9 germ line (..and meiotic) mutations Responsible for inherited diseases
somatic mutations Tumors ? Cell specific and/or tissue alterations TNR instability caused by polymerase slippage. DNA polymerase strand slippage has been proposed as the primary mechanism for instability of TNR. During replication, the TNR units can misalign resulting in an extrahelical DNA loop that increases TNR length if it occurs on the daughter strand (expansion), and decreases TNR length if it occurs on the template strand (deletion). Loop / hairpin structures when not properly repaired (no repair) (reference 43) are incorporated into the nascent strand (expansion, bottom) or skipped (deletion, top). Pol is DNA polymerase. Cell Research (2008) 18:198–213. doi: /cr Features of trinucleotide repeat instability in vivo Irina V Kovtun and Cynthia T McMurray

10 Espansione in regioni non codificanti:
Sindrome da X fragile Causata da un’espansione CGG nel 5’ UTR del gene fragile X mental retardation (FMR1), localizzato sul cromosoma X. Colpisce solo gli uomini. Sintomi:Ritardo mentale-Ritardo dello sviluppo del linguaggio verbale-Iperattività-Sterilità 6-55 ripetizioni-soggetto sano ripetizioni-“premutazione” Oltre 200 ripetizioni- patologia

11 Espansione in regioni non codificanti:
Sindrome da X fragile A livello molecolare: L’espansione CGG nel 5’ UTR del gene causa ipermetilazione della regione richiamando metil DNA binding protein ed enzimi che modificano gli istoni (HDAC) che inibiscono la trascrizione genica

12 Sindrome da X fragile- approcci terapeutici
Espansione in regioni non codificanti: Sindrome da X fragile- approcci terapeutici Agenti demetilanti (5-azadeoxycytidina o 5-aza) Inibitori di HDAC Uso sinergistico dei 2 composti

13 Espansione in regioni non codificanti:
Friedrich Ataxia La mutazione del gene più comune, che codifica per una proteina detta Frataxina, è costituita dalla ripetizione della tripletta GAA situata nel primo introne del gene. La sequenza di queste basi nucleotidiche, che normalmente ha un massimo di 40 triplette negli individui normali, si espande fino ad alcune centinaia nei pazienti malati.
L’effetto è una diminuzione marcata di livello di RNA della Frataxina nonchè della quantità di Frataxina espressa, anche se una minima parte viene comunque prodotta.
La Frataxina è una proteina mitocondriale e agirebbe regolando il flusso del ferro all’interno dei mitocondri stessi.La perdita di frataxina porta ad accumulo di ferro a livello mitocondriale, aumentata suscettibilità allo stress ossidativo, riduzione della fosforilazione ossidativa.

14 Espansione in regioni non codificanti:
Friedrich Ataxia-approcci terapeutici antiossidanti (Idebone. Analogo a catena corta del coenzima Q10) Chelazione del Fe mitocondriale (pyridoxal isonicotynoil hydrazane-PIH, mobilizza selettivamente il Fe mitocondriale.) Modulazione degli istoni (inibitori di HDAC)

15 Gatchel & Zoghbi Nature Rev Genet 2005, 6: 743
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16 Sono proteine espresse in modo ubiquitario, ma presentano disfunzioni solo a livello neurologico.
Sono caratterizzate da una lenta progressione e sono tutte incurabili. Malattie autosomiche, ad eccezione di SBMA che è legata Al cromosoma X. Caratterizzate da fenomeno dell’anticipazione

17 Ross C.A. (1995) Neuron 15:

18 Huntington

19 HD: Htt è localizzata nel citoplasma, assoni, sinapsi e nel nucleo. E’ coinvolta nello shuttling nucleo-citoplasma e nella regolazione trascrizionale. si lega al repressore trascrizionale (REST/NRSF=neuron restrictive silencer factor) e lo sequestra nel citoplasma. NRSF non può agire sul DNA sui NRSE presenti su geni come BDNF.

20 HD: -Ha ruolo nel trasporto assonale,interazioni con vescicole, interagisce con diverse proteine responsabili del trasporto intracellulare ed endocitosi. Regola signalling del calcio

21 Spinal and Bulbar Muscular Atrophy (SBMA) or Kennedy's disease
X-linked disease associated to a CAG repeat expansion in the androgen receptor gene; The CAG repeat is translated to an elongated polyglutamine tract in the N-terminal transactivation domain of the androgen receptor protein. Motoneuronal cell death. - Clinical features: . Motor cranial nerve deficits, amyotrophy, sensory neuropathy - Types of lesion . Gynecomastia, motor neuron loss in the brain stem and spinal cord . Distal axonopathy, atrophy of dorsal root ganglion root - Neuroanatomic distribution of the lesion . Motor nerve nuclei of brain stem . Anterior horn in the spinal cord, dorsal root ganglia

22 Zn mRNA protein polyGln Poletti Front Neuroendocrinol 2004 normal
.....CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG..... mutant .....CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG..... Zn polyGln P PP P P A S P S P P = phosphorylation A = acetylation S = sumoylation Transactivation domain/(AF-1/AF-5) \ ID NLS AF-2 DNA binding domain H12 Hormone binding 5'-UTR Exon Exon 2 Exon 3 Exon 4 Exons 5/6/7/8 3'-UTR (CAG)n Upstream ORF AUG1-UGA AUG2 mRNA protein polyA Poletti Front Neuroendocrinol 2004 22

23 23

24 Spinal and Bulbar Muscular Atrophy (SBMA)
Human X-linked disease the AR gene is on X-chrom - Only males affected - gain-of-function of SBMA AR Females may show few clinical symptoms: --> random X-chromosome inactivation ?? --> effect of testosterone ?? TWO Homozygous females --> NO CLINICAL SYMPTOMS Schmidt et al. Neurology (2002) 59: Tg mouse for SBMA AR transgene randomly integrated in the genome - Only males affected (!!) - gain-of-function of SBMA AR Females DO NOT show clinical symptoms --> random X-chromosome inactivation NOT RELATED --> effect of testosterone Katsuno et al. Neuron (2002) 35: Katsuno et al. Nat Med (2003) 9: 24

25 Cx Disease NO Disease T No symptoms Symptoms 25

26 Malattie da PolyQ: -perché solo nei neuroni? -perché solo in alcune popolazioni neuronali??

27 Malattie da PolyQ - Patologie che insorgono con l’età:
Le proteine mutate acquistano tossicità, il tratto polyQ modifica la conformazione e la funzione provocando (GAIN of FUNCTION) Interazioni anomale tra proteine Modificazione della localizzazione cellulare Tossicità cellulare Suscettibilità al taglio proteolitico

28 Malattie da PolyQ - Patologie che insorgono con l’età:
Proteine misfolded - GAIN OF FUNCTION Formazione di inclusioni intracellulari Alterazioni trascrizionali Sequestro di proteine importanti Hsps Alterazioni dei sistemi degradativi Alterazioni del trasporto assonale Danni mitocondriali

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30 Figure 1. Polyglutamine pathogenesis: a multimodal hypothesis
Figure 1. Polyglutamine pathogenesis: a multimodal hypothesis. Various pathogenic pathways have been suggested. As a simplification, they are depicted here as originating in the cytoplasm, although some of the disease proteins, including huntingtin, might also be located in the nucleus or cycle between the cytoplasm and nucleus. (a) The pathogenic process (blue arrows) begins with the synthesis of a protein with an expanded polyglutamine (polyQ) tract. (b) The expanded polyglutamine tract alters the native conformation of the protein, modulated by the presence of molecular chaperones. (c) At least a fraction of the abnormally folded protein is subjected to lysosomal-dependent proteolysis, and (d) another portion of the abnormal protein is ubiquitinated (Ub) and degraded via the proteasome. (e) Cleavage of the abnormally folded mutant protein produces an N-terminal fragment that favours the aggregation process. (f) The mutant proteins shift, in part, from a monomeric random coil or b-sheet into oligomeric b-sheets and eventually into insoluble aggregates (amyloid fibrils). (g) These might contribute to pathology through abnormal interactions with cellular proteins, or might represent a mechanism for reducing the toxicity of aggregation intermediates. (h) Aggregation intermediates inhibit proteasomal processing. (i) The monomers or oligomers directly activate caspases or disrupt mitochondrial function, leading to indirect activation of caspases. (j) Proto-aggregates translocate into the nucleus (by an unknown mechanism) and (k) recruit specific nuclear factors, co-activators and co-repressors, inhibiting their normal activities and (l) resulting in altered gene transcription (an example is the loss of function of proteins with histone acetyltransferase activity). The pathogenic pathways depicted here suggest a number of potential sites for therapeutic intervention (indicated in red). These include: (1) inhibition of expression of the mutant protein at the level of transcription or translation; (2) facilitation of chaperone function; (3) inhibition of proteolysis; (4) inhibition of aggregation (by enhancement of chaperones or by pharmacological agents such as Congo Red that suppress formation of intermediates or protofibrillar assembly into insoluble aggregates); (5) mitochondrial stabilisation (agents such as creatine that protect against bioenergetic dysfunction); (6) caspase inhibition; (7) inhibition of histone deacetylase (HDAC) activity; and (8) modulation of transcription that is adversely affected by mutant huntingtin. This model is most representative of HD pathogenesis, but also pertains, in part, to other polyglutamine diseases (fig001rmb). Expert Reviews in Molecular Medicine: Accession information: Vol. 5; 22 August 2003 
PDF Polyglutamine pathogenesis: a multimodal hypothesisDobrila D. Rudnicki and Russell L. Margolis 30

31 Meccanismi patologici della malattie polyQ
Misfolding e aggregazione Gli aggregati si formano a livello citoplasmatico e nucleare. Il loro ruolo non è ancora ben chiaro.

32 Fig. 1 Immunohistochemical analysis in the neural tissues from SBMA patients and control cases. In the CNS of SBMA patients, intense diffuse nuclear staining is present in neuronal nuclei of various regions using 1C2 antibody (A, E, F, G, J and L). Diffuse immunostaining of mutant androgen receptor (AR) is present in a web-like pattern in nuclei of anterior horn neurons (A). Diffuse nuclear staining is also observed in posterior horn neurons (E), substantia nigra (F), spinal dorsal root ganglia (G), paravertebral sympathetic ganglia (J) and hypothalamus (L). Some nuclei appear packed with mutant AR. Small or large nuclear inclusions are also stained intensely using 1C2 antibody in anterior horn neurons (B–D), posterior horn neurons (E), the substantia nigra (F) and the hypothalamus (L). Most of the dark brown pigment seen in the neuronal cytoplasm in the substantia nigra (F) is neuromelanin. In addition to nuclear inclusions, occasional neurons exhibit granular structures immunoreactive for 1C2 in the cytoplasm, such as in the anterior horn (D) and hypothalamus (K). In spinal dorsal root ganglia, small or large cytoplasmic inclusions are frequent (H and I). There is no immunoreactivity for 1C2 in the spinal anterior horn cell from the control case (M). Immunopositive nuclear inclusions and diffuse nuclear staining are also present using H280 antibody in the spinal anterior horn cell (N) and spinal dorsal root ganglia (O). Spinal dorsal root ganglia neurons exhibit granular structures immunoreactive for anti-Golgi 58K protein antibody in the cytoplasm in SBMA (P) and a control case (Q). Adapted from: Adachi H et al. Widespread nuclear and cytoplasmic accumulation of mutant androgen receptor in SBMA patients. Brain 2005 (doi: /brain/awh381)

33

34 Atomic force microscopy shows the presence of
fibrillar forms of the purified recombinant AR.Q46 N-terminal fragment expressed in E.Coli Palazzolo I et al., JSB (2008) 108:

35 GFP in pEGFP-C1 GFP-AR.Q0 GFP-AR.Q22 GFP-AR.Q48 NH2 COOH DNA HORMONE
HINGE N - terminal NH2 GFP

36 - T + T Aggregate formation in immortalized motorneurons expressing
androgen receptor with an elongated polyglutamine tract GFP-AR.Q0 GFP-AR.Q22 GFP-AR.Q48 - T + T Simeoni et al. Hum Mol Genet 2000

37 Effects of testosterone on ARpolyQ solubility in SBMA
Rusmini et al. Neurobiol Ag 2007

38

39 Redistribution of GFP-AR.Q48 aggregates after ligand removal
courtesy by Michael A. Mancini, Dpt. Cell Biology Baylor College of Medicine, Houston, Tx, USA Aggregates induced by 2h T-treatment

40 Aggregates of Full-lenght AR.Q48
Neuropil aggregates in immortalized motorneurons expressing androgen receptor with an elongated polyglutamine tract Aggregates of Full-lenght AR.Q48 29 cytoplasmic 0.3 neuropil 6 nuclear Simeoni et al. Hum Mol Genet 2000 NSC34/AR.Q NSC34/AR.Q22 NSC34/AR.Q46

41 NSC34/GFP-AR.Q0/mtBFP NSC34/GFP-AR.Q22/mtBFP NSC34/GFP-AR.Q48/mtBFP
Piccioni et al. FASEB J 2002

42 Mitochondria accumulation in immortalized motorneurons
bearing aggregates in cell processes Poletti Front Neuroendocrinol 2004

43 Neuropil aggregates in immortalized motor neurons
expressing SBMA androgen receptor Red = AR.Q48 Green = tubulin Blue = kinesin

44 Goldstein L.S. Do disorders of movement cause movement disorders and dementia?
Neuron (2003) 40:415-25

45 SBMA AR

46 SBMA AR + TESTOSTERONE Cytoplasmic inclusions toxicity debated

47 SBMA AR + TESTOSTERONE Neuropil inclusions axonal dysfunctions a) Migration from cell soma ? b) "In situ" formation ?

48 SBMA AR + TESTOSTERONE Neuropil inclusions axonal dysfunctions Neurite damage fast axonal transport alteration

49 SBMA AR + TESTOSTERONE Neuropil inclusions axonal dysfunctions Axonal degeneration

50 SBMA AR + TESTOSTERONE Neuropil inclusions Cell death

51 Meccanismi patologici della malattie polyQ
Misfolding e aggregazione Gli aggregati si formano a livello citoplasmatico e nucleare. Il loro ruolo non è ancora ben chiaro. 2. Modifiche post-traduzionali-Taglio proteolitico Produzione di corti frammenti tossici.

52 TRUNCATED FORMS of the ANDROGEN RECEPTOR AR16HA e AR112HA
Caspase-3 cleavage site Caspase-3 cleavage product of AR identified in spinal cord motoneurons of SBMA patients NH2 COOH DBD polyQ transactivation domain hormone binding domain polyQ = 16 polyQ = 112 AR16HA AR112HA Courtesy by DE Merry, TJU, USA

53 Intranuclear inclusion in immortalized motoneurons expressing AR
Intranuclear inclusion in immortalized motoneurons expressing AR.Q112  HA Mk Mock Q16 Q112 <-- AR16.DHA AR112.DHA monomer dimer <-- AR112.DHA aggregates <-- Detergent (SDS) resistant intracellular aggregates NT pCDNA3+pUC AR_∆16+pUC AR_∆112+pUC pCDNA3+Hsp70 AR_∆16+Hsp70 AR_∆112+Hsp70 The GFPu protein is a modification of an unstable form of GFP, targeted to the proteasome system by fusion with the following peptide: Ala-Cys-Lys-Asn-Trp-Phe-Ser-Ser-Leu-Ser-His-Phe-Val-Ile-Hys-Leu Courtesy by R KOPITO, USA AR.Q112  HA DAPI Merge 53

54 Meccanismi patologici della malattie polyQ
Misfolding e aggregazione Gli aggregati si formano a livello citoplasmatico e nucleare. Il loro ruolo non è ancora ben chiaro. 2. Modifiche post-traduzionali-Taglio proteolitico Produzione di corti frammenti tossici. 3. Localizzazione nucleare della Proteina tossica Alterazione dell’espressione genica e dell’organizzazione nucleare. 4. Hsp-andamento bifasico. Alta espressione nella fase Iniziale, riduzione nelle fasi finali.

55 Meccanismi patologici della malattie polyQ
Misfolding e aggregazione Gli aggregati si formano a livello citoplasmatico e nucleare. Il loro ruolo non è ancora ben chiaro. 2. Modifiche post-traduzionali-Taglio proteolitico Produzione di corti frammenti tossici. 3. Localizzazione nucleare della Proteina tossica Alterazione dell’espressione genica e dell’organizzazione nucleare. 4. Hsp-andamento bifasico. Alta espressione nella fase Iniziale, riduzione nelle fasi finali.

56 HSP70 sequestration in SBMA AR aggregates

57 Meccanismi patologici della malattie polyQ
Misfolding e aggregazione Gli aggregati si formano a livello citoplasmatico e nucleare. Il loro ruolo non è ancora ben chiaro. 2. Modifiche post-traduzionali-Taglio proteolitico Produzione di corti frammenti tossici. 3. Localizzazione nucleare della Proteina tossica Alterazione dell’espressione genica e dell’organizzazione nucleare. 4. Hsp-andamento bifasico. Alta espressione nella fase Iniziale, riduzione nelle fasi finali. 5. Alterazione dei sistemi degradativi: proteasome e autofagia

58 Molecular pathogenesis of the polyglutamine diseases and the protein quality control system
Naiki, H. et al. J Biochem : ; doi: /jb/mvp119 Copyright restrictions may apply.

59 Ubiquitin positive - AR aggregates
Misfolded proteins Polyubiquitination Proteasomal Degradation Goldberg AL Protein degradation and protection against misfolded or damaged proteins Nature 426, (2003) Nature Medicine 10, S10 - S17 (2004) Published online: 01 July 2004; | doi: /nm1066 Protein aggregation and neurodegenerative diseaseChristopher A Ross & Michelle A Poirier Figure 3. Flowchart for therapeutic intervention in a hypothetical several-step pathway of protein aggregation.An initiating event in aggregation may be covalent modification of the disease protein, for example by cleavage or phosphorylation, facilitating conversion of the protein to an abnormal conformation. Oligomeric (globular) intermediates may form, and then protofibrillar structures are assembled. Amyloid fibers can then form, possibly through association of protofibrillar intermediates, resulting in aggregates or inclusions visible in the light microscope. The intermediate species are hypothesized to be more toxic than either the precursor protein or the aggresomes and inclusions. Inhibition early in the pathway would be beneficial to the cell, because it may prevent the formation of potentially toxic oligomeric or other intermediates. (In a model with linear addition and no oligomeric intermediates, the process of polymerization itself would be presumed to be toxic.) By contrast, inhibition at later stages could be detrimental, because it may result in accumulation of toxic intermediates. If inhibitors could be developed that would act at the intermediate steps, they could help identify which intermediate is the toxic species. This model is based on a very hypothetical pathway for polyglutamine aggregation51, 57, and the details are subject to change; however, the concept may be generally applicable. 59

60 Taylor et al. Science 291:1991, 2002 60

61 Effects of testosterone on proteasome
functions in SBMA misfolded proteins GFP DEGRON SIGNAL FOR UPP A-C-K-N-W-P-S-S-L-S-H-P-V-I-H-L Ub GFPu degradation GFPu accumulation ARpolyQ Rusmini et al. Neurobiol Ag 2007 61

62 Certain key neuronal proteins may misfold
Certain key neuronal proteins may misfold. These misfolded proteins can become ubiquitinated (red circles) and degraded by the proteasome. However, these proteins may be poor substrates for the proteasome, and instead will accumulate in the cytoplasm, making microaggregates and possibly leading to cell death. Basal autophagy can keep the levels of these proteins low enough to prevent toxic effects by sequestering them inside autophagosomes that deliver them to the lysosome for subsequent degradation. The misfolded proteins may also form large aggregates or inclusion bodies that can induce an autophagic response. These large aggregates may be more protective than harmful. Nature 441, (15 June 2006) | doi: /441819° Neurodegeneration: Good riddance to bad rubbish Daniel J. Klionsky1 62 62

63 Quali approcci terapeutici????????
1.Fattori neurotrofici (infusione, vettori virali,cell delivery) BDNF, VEGF 2.Trapianti cellulari per ripristinare i circuiti neuronali esperimenti in HD trapianto di cellule fetali striatali,analisi post-mortem ha rivelato che le cellule si erano impiantate ma non si sono riscontrati benefici per la malattia. 3.Somministrazione di RNA antisenso per ridurre i livelli Trascrizionali, per silenziare solo l’allele polimorfico.

64 Quali approcci terapeutici????????
4. Inibitori delle Caspasi zVAD-fmk minociclina antibiotico inibitore della caspasi 1 e 3 5. Overespressione di fattori trascrizionali 6. Inibitori di HDAC Fenilbutirrato, acido valproico Il sequestro di fattori trascrizionali ad azione acetil-trasferasica posso portare ad alterazione dell’attività trascrizionale alterando i livelli di acetilazione degli istoni. Questo effetto può essere controbilanciato con l’inibizione dell’attività de-acetilasica.

65 Quali approcci terapeutici????????
7. degradazione proteica via UPS e autofagia e inibizione dell’aggregazione Trealosio- Congo Red, Litio, 17-AAG, overespressione di Chaperone

66

67 Litio inibisce inositolo monofosfatasi e porta alla deplezione di inositolo libero e di conseguenza di inositolo tri-fosfato (IP3)

68 native protein unfolding intermediate aggregate
HSP sHSPs proteasomal degradation ADP ATP stress Modified from Haslbeck and Buchner (2002)

69 There are two types of expression patterns for sHsps.
HspB1, HspB5, HspB6, and HspB8 all show ubiquitous expression with varying concentrations in the different tissues. The tissue distribution of HspB2, HspB3, and HspB7 is restricted largely to heart and muscle. HspB4 is found in the eye lens. HspB9 is expressed specifically in testis (notably in the spermatogenic cells from late pachytene spermatocyte stage till elongate spermatid stage). HspB10 is located exclusively as a major component in sperm cell tails. Hu et al. J Neurosci Res, Feb 07, DOI:

70 Moreover, HspB8... Lys41 mutations have been linked to either distal motor neurophaty or CMT_2L; has been found in senile plaques of Alzheimer’s disease brains (Wilhelmus et al, 2006) inhibits in vitro aggregation of amyloid- protein (Wilhelmus et al, 2006)

71 Effects of HspB8 on the SBMA ARpolyQ aggregation and on proteasome functions
ARQ46 HspB8 T [10-8] AR actin HspB8 YFPu ARQ23 ARQ46 T [10-8]M YFPu contains the CL1 an hydrophobic peptide that generates a misfolded domain

72 Targeting toxic proteins for turnover
Figureﾊ1.ﾊAction of a heat-shock protein 90 inhibitor in the polyglutamine-repeat disease SBMA.SBMA is caused by polyglutamine-repeat expansions in the androgen receptor, which result in the formation of neuronal aggregates of this protein. A soluble, nontoxic form of this protein probably exists in equilibrium with a soluble toxic protein. The soluble form shifts to an oligomeric or protofibrillar state before being assembled into a more orderly aggregate structure. The adoption of an altered conformation by the protein renders it resistant to protein degradation, and is thus central to polyglutamine pathogenicity. Waza et al. report that 17-allylamino geldanamycin (17-AAG) prevents neurotoxicity in a mouse model of SBMA by promoting the degradation of polyglutamine-expanded androgen receptor. Treatment with 17-AAG caused the dissociation of p23 from the Hsp90−androgen receptor complex. Although Waza et al. suggest that 17-AAG drives the turnover of polyglutamine-expanded androgen receptor by a proteasome-dependent pathway, the extent to which the proteasome alone can degrade a polyglutamine-expanded protein remains controversial. Nature Medicine 11, (2005) doi: /nm Targeting toxic proteins for turnover Albert R La Spada & Patrick Weyd 72

73 17AAG increases the degradation of the misfolded SBMA ARpolyQ
17AAG: Hsp90 inhibitor / Hsp70 inducer AR YFPu actin EtOH / EtOH EtOH / T T / T 17AAG / T 17AAG / 17AAG+T DMSO MG132 NT T : 10 nM 17AAG : 330 nM MG132 : 10 M

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