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1.
Acta Neuropathol ; 136(6): 919-938, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30140941

RESUMO

The deposition of pathologic misfolded proteins in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia and amyotrophic lateral sclerosis is hypothesized to burden protein homeostatic (proteostatic) machinery, potentially leading to insufficient capacity to maintain the proteome. This hypothesis has been supported by previous work in our laboratory, as evidenced by the perturbation of cytosolic protein solubility in response to amyloid plaques in a mouse model of Alzheimer's amyloidosis. In the current study, we demonstrate changes in proteome solubility are a common pathology to mouse models of neurodegenerative disease. Pathological accumulations of misfolded tau, α-synuclein and mutant superoxide dismutase 1 in CNS tissues of transgenic mice were associated with changes in the solubility of hundreds of CNS proteins in each model. We observed that changes in proteome solubility were progressive and, using the rTg4510 model of inducible tau pathology, demonstrated that these changes were dependent upon sustained expression of the primary pathologic protein. In all of the models examined, changes in proteome solubility were robust, easily detected, and provided a sensitive indicator of proteostatic disruption. Interestingly, a subset of the proteins that display a shift towards insolubility were common between these different models, suggesting that a specific subset of the proteome is vulnerable to proteostatic disruption. Overall, our data suggest that neurodegenerative proteinopathies modeled in mice impose a burden on the proteostatic network that diminishes the ability of neural cells to prevent aberrant conformational changes that alter the solubility of hundreds of abundant cellular proteins.


Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Doenças Neurodegenerativas/patologia , Emaranhados Neurofibrilares/patologia , Proteoma/metabolismo , Fatores Etários , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Doenças Neurodegenerativas/genética , Emaranhados Neurofibrilares/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Dobramento de Proteína , Proteoma/genética , Solubilidade , Espectrometria de Massas em Tandem , alfa-Sinucleína/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo
2.
Mol Ther ; 23(1): 53-62, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25228069

RESUMO

The architecture of the spinal cord makes efficient delivery of recombinant adeno-associated virus (rAAV) vectors throughout the neuraxis challenging. We describe a paradigm in which small amounts of virus delivered intraspinally to newborn mice result in robust rAAV-mediated transgene expression in the spinal cord. We compared the efficacy of rAAV2/1, 2/5, 2/8, and 2/9 encoding EGFP delivered to the hindlimb muscle (IM), cisterna magna (ICM), or lumbar spinal cord (IS) of neonatal pups. IS injection of all four capsids resulted in robust transduction of the spinal cord with rAAV2/5, 2/8, and 2/9 vectors appearing to be transported to brain. ICM injection resulted in widespread expression of EGFP in the brain, and upper spinal cord. IM injection resulted in robust muscle expression, with only rAAV2/8 and 2/9 transducing spinal motor and sensory neurons. As proof of concept, we use the IS paradigm to express murine Interleukin (IL)-10 in the spinal cord of the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis. We show that expression of IL-10 in the spinal axis of SOD1-G93A mice altered the immune milieu and significantly prolonged survival. These data establish an efficient paradigm for somatic transgene delivery of therapeutic biologics to the spinal cord of mice.


Assuntos
Esclerose Lateral Amiotrófica/terapia , Encéfalo/metabolismo , Dependovirus/genética , Terapia Genética/métodos , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Capsídeo/metabolismo , Cisterna Magna/metabolismo , Cisterna Magna/patologia , Dependovirus/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Injeções Espinhais , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Transdução Genética
3.
J Neurochem ; 133(3): 452-64, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25557022

RESUMO

There has been great interest in enhancing endogenous protein maintenance pathways such as the heat-shock chaperone response, as it is postulated that enhancing clearance of misfolded proteins could have beneficial disease modifying effects in amyotrophic lateral sclerosis and other neurodegenerative disorders. In cultured cell models of mutant SOD1 aggregation, co-expression of αB-crystallin (αB-crys) has been shown to inhibit the formation of detergent-insoluble forms of mutant protein. Here, we describe the generation of a new line of transgenic mice that express αB-crys at > 6-fold the normal level in spinal cord, with robust increases in immunoreactivity throughout the spinal cord grey matter and, specifically, in spinal motor neurons. Surprisingly, spinal cords of mice expressing αB-crys alone contained 20% more motor neurons per section than littermate controls. Raising αB-crys by these levels in mice transgenic for either G93A or L126Z mutant SOD1 had no effect on the age at which paralysis developed. In the G93A mice, which showed the most robust degree of motor neuron loss, the number of these cells declined by the same proportion as in mice expressing the mutant SOD1 alone. In paralyzed bigenic mice, the levels of detergent-insoluble, misfolded, mutant SOD1 were similar to those of mice expressing mutant SOD1 alone. These findings indicate that raising the levels of αB-crys in spinal motor neurons by 6-fold does not produce the therapeutic effects predicted by cell culture models of mutant SOD1 aggregation. Enhancing the protein chaperone function may present a therapeutic approach to amyotrophic lateral sclerosis caused by mutations in SOD1, and other neurodegenerative disorders characterized by cytosolic protein aggregation. Previous studies in cell models suggested that the chaperone known as αB-crystallin (αB-crys) can prevent mutant SOD1 aggregation. We report that transgenic expression of αB-crys at > 6-fold the normal level in spinal cords of mice expressing mutant SOD1 produces no therapeutic benefit.


Assuntos
Neurônios Motores/metabolismo , Proteínas Mutantes/biossíntese , Paralisia/metabolismo , Agregação Patológica de Proteínas/metabolismo , Superóxido Dismutase , Cadeia B de alfa-Cristalina/biossíntese , Animais , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mutantes/genética , Paralisia/genética , Paralisia/prevenção & controle , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/prevenção & controle , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Cadeia B de alfa-Cristalina/genética
4.
Nat Commun ; 12(1): 2770, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986266

RESUMO

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.


Assuntos
Sistemas CRISPR-Cas/genética , Hipercolesterolemia/genética , Lipossomos/farmacologia , Pré-Albumina/metabolismo , Ativação Transcricional/genética , Animais , Linhagem Celular , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , Células HEK293 , Humanos , Hipercolesterolemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas , Pré-Albumina/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
5.
G3 (Bethesda) ; 9(3): 719-728, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30622123

RESUMO

Mutations in more than 80 different positions in superoxide dismutase 1 (SOD1) have been associated with amyotrophic lateral sclerosis (fALS). There is substantial evidence that a common consequence of these mutations is to induce the protein to misfold and aggregate. How these mutations perturb native structure to heighten the propensity to misfold and aggregate is unclear. In the present study, we have mutagenized Glu residues at positions 40 and 133 that are involved in stabilizing the ß-barrel structure of the native protein and a critical Zn binding domain, respectively, to examine how specific mutations may cause SOD1 misfolding and aggregation. Mutations associated with ALS as well as experimental mutations were introduced into these positions. We used an assay in which mutant SOD1 was fused to yellow fluorescent protein (SOD1:YFP) to visualize the formation of cytosolic inclusions by mutant SOD1. We then used existing structural data on SOD1, to predict how different mutations might alter local 3D conformation. Our findings reveal an association between mutant SOD1 aggregation and amino acid substitutions that are predicted to introduce steric strain, sometimes subtly, in the 3D conformation of the peptide backbone.


Assuntos
Esclerose Lateral Amiotrófica/genética , Mutação , Agregação Patológica de Proteínas , Dobramento de Proteína , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/enzimologia , Animais , Células CHO , Cricetulus , Humanos , Conformação Proteica , Superóxido Dismutase-1/metabolismo
6.
PLoS One ; 13(11): e0206751, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30399166

RESUMO

Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis (fALS) induce the protein to misfold and aggregate. Missense mutations at more than 80 different amino acid positions have been associated with disease. How these mutations heighten the propensity of SOD1 to misfold and aggregate is unclear. With so many mutations, it is possible that more than one mechanism of aggregation may be involved. Of many possible mechanisms to explain heightened aggregation, one that has been suggested is that mutations that eliminate charged amino acids could diminish repulsive forces that would inhibit aberrant protein:protein interactions. Mutations at twenty-one charged residues in SOD1 have been associated with fALS, but of the 11 Lys residues in the protein, only 1 has been identified as mutated in ALS patients. Here, we examined whether loss of positively charged surface Lys residues in SOD1 would induce misfolding and formation of intracellular inclusions. We mutated four different Lys residues (K30, K36, K75, K91) in SOD1 that are not particularly well conserved, and expressed these variants as fusion proteins with yellow fluorescent protein (YFP) to assess inclusion formation. We also assessed whether these mutations induced binding to a conformation-restricted SOD1 antibody, designated C4F6, which recognizes non-natively folded protein. Although we observed some mutations to cause enhanced C4F6 binding, we did not observe that mutations that reduce charge at these positions caused the protein to form intracellular inclusions. Our findings may have implications for the low frequency of mutations at Lys residues SOD1 in ALS patients.


Assuntos
Corpos de Inclusão/metabolismo , Mutação , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CHO , Cricetulus , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína
7.
J Comp Neurol ; 524(14): 2740-52, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-26878116

RESUMO

Mutations in the MATR3 gene encoding the nucleotide binding protein Matrin 3 have recently been identified as causing a subset of familial amyotrophic lateral sclerosis (fALS) and more rarely causing distal myopathy. Translating the identification of MATR3 mutations into an understanding of disease pathogenesis and the creation of mouse models requires a complete understanding of normal Matrin 3 levels and distribution in vivo. Consequently, we examined the levels of murine Matrin 3 in body tissues and regions of the central nervous system (CNS). We observed a significant degree of variability in Matrin 3 protein levels among different tissues of adult animals, with the highest levels found in reproductive organs and the lowest in muscle. Within the adult CNS, Matrin 3 levels were lowest in spinal cord. Further, we found that Matrin 3 declines significantly in CNS through early development and young adulthood before stabilizing. As previously reported, antibodies to Matrin 3 primarily stain nuclei, but the intensity of staining was not uniform in all nuclei. The low levels of Matrin 3 in spinal cord and muscle could mean that that these tissues are particularly vulnerable to alterations in Matrin 3 function. Our study is the first to characterize endogenous Matrin 3 in rodents across the lifespan, providing the groundwork for deciphering disease mechanisms and developing mouse models of MATR3-linked ALS. J. Comp. Neurol. 524:2740-2752, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Envelhecimento/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteínas Associadas à Matriz Nuclear/biossíntese , Proteínas de Ligação a RNA/biossíntese , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL
8.
BMC Med Genomics ; 8: 5, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25889064

RESUMO

BACKGROUND: Adoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations. METHODS: In this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing). RESULTS: Cross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%. CONCLUSIONS: Reproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy.


Assuntos
Técnicas de Laboratório Clínico/normas , Perfilação da Expressão Gênica/métodos , Genoma Humano , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fixação de Tecidos/métodos , Análise Mutacional de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Perda de Heterozigosidade , Masculino , Mutação , Neoplasias/metabolismo , Inclusão em Parafina , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA
9.
PLoS One ; 8(12): e83981, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24391857

RESUMO

BACKGROUND: By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures. CONCLUSIONS/SIGNIFICANCE: Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells.


Assuntos
Proteínas de Bactérias/metabolismo , Corpos de Inclusão/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas Mutantes/metabolismo , Dobramento de Proteína , Superóxido Dismutase/fisiologia , Animais , Proteínas de Bactérias/genética , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Multimerização Proteica , Saponinas/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase-1 , Proteína Vermelha Fluorescente
10.
Mol Neurodegener ; 8: 46, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24341866

RESUMO

Mutations in the gene encoding superoxide dismutase 1 (SOD1) account for about 20% of the cases of familial amyotrophic lateral sclerosis (fALS). It is well established that mutations in SOD1, associated with fALS, heighten the propensity of the protein to misfold and aggregate. Although aggregation appears to be a factor in the toxicity of mutant SOD1s, the precise nature of this toxicity has not been elucidated. A number of other studies have now firmly established that raising the levels of wild-type (WT) human SOD1 (hSOD1) proteins can in some manner augment the toxicity of mutant hSOD1 proteins. However, a recent study demonstrated that raising the levels of WT-hSOD1 did not affect disease in mice that harbor a mouse Sod1 gene (mSod1) encoding a well characterized fALS mutation (G86R). In the present study, we sought a potential explanation for the differing effects with WT-hSOD1 on the toxicity of mutant hSOD1 versus mutant mSod1. In the cell culture models used here, we observe poor interactions between WT-hSOD1 and misfolded G86R-mSod1, possibly explaining why over-expression of WT-hSOD1 does not synergize with mutant mSod1 to accelerate the course of the disease in mice.


Assuntos
Esclerose Lateral Amiotrófica , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Dobramento de Proteína , Superóxido Dismutase-1 , Transfecção
11.
PLoS One ; 8(6): e67680, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23825679

RESUMO

Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24-84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.


Assuntos
Encéfalo/metabolismo , Capsídeo/metabolismo , Dependovirus/classificação , Dependovirus/genética , Sorotipagem , Transdução Genética/métodos , Animais , Animais Recém-Nascidos , Astrócitos/virologia , Encéfalo/citologia , Encéfalo/virologia , Proteínas de Fluorescência Verde/genética , Injeções , Camundongos , Fatores de Tempo , Tropismo Viral
12.
PLoS One ; 7(12): e50750, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23236391

RESUMO

BACKGROUND: N-terminal fragments of mutant huntingtin (htt) that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1), form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD). These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments. RESULTS: Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like), were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1. CONCLUSIONS: Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/metabolismo , Núcleo Celular/genética , Citoplasma/genética , Células HEK293 , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/genética , Proteólise
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