Specific inhibition of pathological prion protein accumulation by small interfering RNAs

Daude, Nathalie; Marella, Mathieu; Chabry, Joëlle

doi:10.1242/jcs.00494

Cited by 95 publications

(69 citation statements)

References 28 publications

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“…These include porphyrins (26,27), Congo red and its derivatives (28)(29)(30), acridine and phenothiazine derivatives (17,31,32), heparan sulfate (33), aminoglycan, and polyamines (34,35). Simultaneously, various technological developments have been reported including structure-based drug design (36) followed by the structure-activity relationship study (37), small interfering RNA (38), library screening (18), high-throughput screening (39), chimeric ligand approach (40), and so on. Although strategies for drug discovery used in these studies have a broad spectrum from empirical to rational preponderance, there has been no report on the residue-specific evidences for the binding regions of antiprion compounds.…”

Section: Discussionmentioning

confidence: 99%

Hot spots in prion protein for pathogenic conversion

Kuwata

Nishida

Matsumoto

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

174

225

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Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrP C ) to the scrapie form (PrP Sc ) is still unknown. Here we discovered a chemical chaperone to stabilize the PrP C conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a ''pocket'' created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced PrP Sc . Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of PrP C , will open the way to the development of novel anti-prion drugs.anti-prion compound ͉ binding sites ͉ chemical chaperone ͉ dynamicsbased drug discovery ͉ transmissible spongiform encephalopathy

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Section: Discussionmentioning

confidence: 99%

Hot spots in prion protein for pathogenic conversion

Kuwata

Nishida

Matsumoto

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

174

225

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“…siRNAs corresponding to the murine Prnp gene from codon 392 to 410 (33) were synthesized by Dharmacon RNA Technologies (Lafayette, CO) and were supplied preduplexed. The sequences of the siRNAs are detailed in SI Methods.…”

Section: Methodsmentioning

confidence: 99%

Cellular prion protein regulates β-secretase cleavage of the Alzheimer's amyloid precursor protein

Parkin

Watt²,

Hussain³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

255

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Proteolytic processing of the amyloid precursor protein (APP) by ␤-secretase, ␤-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid ␤ (A␤) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP C ), the causative agent of the transmissible spongiform encephalopathies such as CreutzfeldtJakob disease in humans, remains enigmatic. Because both APP and PrP C are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP C in the proteolytic processing of APP. Cellular overexpression of PrP C inhibited the ␤-secretase cleavage of APP and reduced A␤ formation. Conversely, depletion of PrP C in mouse N2a cells by siRNA led to an increase in A␤ peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapieinfected mice, A␤ levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the ␤-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP C on the ␤-secretase cleavage of APP required the localization of PrP C to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP C via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic A␤ is regulated by PrP C and may have implications for both Alzheimer's and prion diseases.lipid raft ͉ proteolysis ͉ scrapie ͉ glycosaminoglycan A lzheimer's disease (AD) is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles within the afflicted brain. The major constituents of senile plaques are the amyloid ␤ (A␤) peptides, which are derived from the proteolytic processing of the amyloid precursor protein (APP) (1). In the amyloidogenic pathway, ␤-secretase cleavage of APP yields a soluble N-terminal fragment sAPP␤, along with a short membrane-bound C-terminal fragment that is subsequently cleaved by ␥-secretase to release the A␤ peptides. In the alternative, nonamyloidogenic pathway, ␣-secretase cleaves APP within the A␤ sequence, thus precluding the formation of A␤, and releases a soluble N-terminal fragment sAPP␣. The transmembrane aspartyl protease, ␤-site APP cleaving enzyme (BACE1), has been identified as ␤-secretase (2), members of the ADAM (a disintegrin and metalloprotease) family, particularly ADAM10 and ADAM17, are responsible for ␣-secretase cleavage (3), while a complex of at least four proteins, the presenilins, nicastrin, Aph-1, and Pen-2, constitutes the ␥-secretase (2).The prion protein (PrP) is the causative agent of the transmissible spongiform encephalopathies (TSEs) that include CreutzfeldtJakob disease (CJD), Gerstmann-Scheinker-Straussler (GSS) disease, kuru and fatal familial insomnia in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep (4). In these diseases, the normal ce...

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“…This siRNA was able to silence PrP C expression in a human neuroblastoma cell culture (SK-SY5Y), since after the siRNA transfection, cells did not show presence of PrP C 24 h post-transfection and throughout 96 h, which was the period of time of analysis (Figure 3), demonstrating that the designed regiones, por ejemplo, los codones 108 al 114 y del 171 al 177 de la PrP C humana (26) y el codón 392 al 410 de la PrP C de ratón, y también son capaces de reducir la expresión de PrP C (9) , lo que nos indica que el silenciamiento de la PrP C se puede lograr mediante siRNAs dirigidos contra diferentes blancos en el mRNA y que la misma estrategia podría usarse para el control de enfermedades priónicas en animales exóticos, y reducir la posibilidad de transmisión entre individuos.…”

Section: Discussionunclassified

“…Para llevar a cabo la detección de la PrP C por inmunohistoquímica (IHC), diferentes células como NIE-115 (neuroblastoma de ratón), SK-SY5Y (neuroblastoma humano), células de tipo monocitomacrófago humanas GCT, U-937, J-774 y P-338 y la línea celular Vero, proveniente de riñón de mono, (persistently prion-infected) cells have been cured (9) . The siRNAs are efficient against all PrP Sc strains (10) ; however, a mechanism that inserts these molecules in the target cell is lacking.…”

Section: Detección De La Proteína Priónica Celular Mediante Inmunohisunclassified

“…En cultivos celulares como N2a y GT-1 se ha logrado silenciar el gen PRNP previniendo así la formación de PrP Sc (8) . Usando un siRNA complementario a los codones 392 al 410 de la PrP C murina se han curado células N2aS12sc+ infectadas persistentemente con priones (9) . Los siRNAs son eficaces contra todas las cepas de PrP Sc (10) , sin embargo, se carece de un mecanismo que inserte estas moléculas en la célula blanco.…”

Section: Introductionunclassified

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El silenciamiento de la proteína priónica celular (PrPC) mediante RNA de interferencia (siRNA) reduce la infección por HSV-1 y HSV-2 en células SK-SY5Y

Ortega-Soto¹,

Arellano-Anaya²,

Castañeda-Colín³

et al. 2018

Rev. Mex. Cienc. Pecu.

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RESUMENLas encefalopatías espongiformes transmisibles (EETs) son enfermedades neurodegenerativas fatales que afectan a humanos y ciertas especies animales. La hipótesis más aceptada indica que el agente infeccioso, denotado como prion y compuesto principalmente por la Proteína Priónica Scrapie (PrP Sc ), corresponde a una conformación anormal de una proteína codificada por el huésped denominada Proteína Priónica Celular (PrP C ), cuya función es aún desconocida; sin embargo, la expresión ubicua de PrP C así como su elevado grado de conservación entre especies, sugieren un papel importante para esta proteína. En este trabajo se detectó a la PrP C en diferentes tipos celulares incluyendo un cultivo primario de células de peces (Tilapia, Oreochromis spp.). Además, basándonos en la secuencia de la PrP C humana, se diseñó un RNA de interferencia (siRNA) con el fin de silenciar el gen PRNP en células neuronales SK-SY5Y. El siRNA diseñado inhibió la expresión de PrP C a lo largo de las 96 h post-transfección y las células silenciadas fueron menos susceptibles a la infección por HSV-1 y HSV-2, en comparación con células no transfectadas con el siRNA. PALABRAS CLAVE: Proteína Priónica, PrP C , siRNA, HSV-1, HSV-2. ABSTRACTThe transmissible spongiform encephalopathies (TSEs) are neurodegenerative and fatal diseases, affecting humans and some other animal species. The most accepted hypothesis suggests that the infectious agent, named "prion", is composed mainly by the prion protein scrapie (PrP Sc ) and it corresponds to an abnormal conformation of a host encoded protein, the cellular prion protein (PrP C ), which function is still unknown. However, the ubiquitous expression of PrP C and its highly conserved presence among different species suggests it has a very important role in cell functions. In this work, the PrP C in different cell types, including a primary fish cell culture (Oreochromis spp.), was detected. In addition, based on the human PrP C sequence, we designed a short interfering RNA (siRNA) to silence the PRNP gen in neuronal SK-SY5Y cells. The designed siRNA inhibited the PrP C expression along 96 hours posttransfection and the silenced cells were less susceptible to HSV-1 and HSV-2 infections, in comparison to non siRNA-transfected cells.

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Specific inhibition of pathological prion protein accumulation by small interfering RNAs

Cited by 95 publications

References 28 publications

Hot spots in prion protein for pathogenic conversion

Hot spots in prion protein for pathogenic conversion

Cellular prion protein regulates β-secretase cleavage of the Alzheimer's amyloid precursor protein

El silenciamiento de la proteína priónica celular (PrPC) mediante RNA de interferencia (siRNA) reduce la infección por HSV-1 y HSV-2 en células SK-SY5Y

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