RESUMO
Human genome-wide association studies have identified FAN1 and several DNA mismatch repair (MMR) genes as modifiers of Huntington's disease age of onset. In animal models, FAN1 prevents somatic expansion of CAG triplet repeats, whereas MMR proteins promote this process. To understand the molecular basis of these opposing effects, we evaluated FAN1 nuclease function on DNA extrahelical extrusions that represent key intermediates in triplet repeat expansion. Here, we describe a strand-directed, extrusion-provoked nuclease function of FAN1 that is activated by RFC, PCNA, and ATP at physiological ionic strength. Activation of FAN1 in this manner results in DNA cleavage in the vicinity of triplet repeat extrahelical extrusions thereby leading to their removal in human cell extracts. The role of PCNA and RFC is to confer strand directionality to the FAN1 nuclease, and this reaction requires a physical interaction between PCNA and FAN1. Using cell extracts, we show that FAN1-dependent CAG extrusion removal relies on a very short patch excision-repair mechanism that competes with MutSß-dependent MMR which is characterized by longer excision tracts. These results provide a mechanistic basis for the role of FAN1 in preventing repeat expansion and could explain the antagonistic effects of MMR and FAN1 in disease onset/progression.
Assuntos
Estudo de Associação Genômica Ampla , Repetições de Trinucleotídeos , Humanos , Extratos Celulares , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Enzimas Multifuncionais/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Expansão das Repetições de TrinucleotídeosRESUMO
The G98R mutation in αA-crystallin is associated with the onset of presenile cataract and is characterized biochemically by an increased oligomeric mass, altered chaperone function, and loss of structural stability over time. Thus, far, it is not known whether the inherent instability caused by gain-of-charge mutation could be rescued by a compensatory loss of charge mutation elsewhere on the protein. To answer this question, we investigated whether αA-G98R-mediated instability could be rescued through suppressor mutations by introducing site-specific "compensatory" mutations in αA-G98R-crystallin, αA-R21Q/G98R, αA-G98R/R116C, and αA-R157Q/G98R. The recombinant proteins were expressed, purified, characterized, and evaluated by circular dichroism (CD), intrinsic fluorescence, and bis-ANS-binding studies. Chaperone-like activities of recombinant proteins were assessed using alcohol dehydrogenase (ADH) and insulin as unfolding substrates. Far-UV CD studies revealed an increased α-helical content in αA-G98R in comparison to αA-WT, αA-R21Q, R157Q, and the double mutants, αA-R21Q/G98R, and αA-R157Q/G98R. Compared to αA-WT, αA-R21Q, and αA-G98R, the double mutants showed an increased intrinsic tryptophan fluorescence, whereas the highest hydrophobicity (bis-ANS-binding) was shown by αA-G98R. Introduction of a second mutation in αA-G98R reduced its bis-ANS-binding activity. Both αA-R21Q/G98R and αA-R157Q/G98R showed greater chaperone-like activity against ADH aggregation than αA-G98R. However, among the three G98R mutants, only αA-R21Q/G98R protected ARPE-19 cells from H2O2-induced cytotoxicity. These results suggest that the lost chaperone-like activity of αA-G98R-crystallin can be rescued by another targeted mutation and that substitution of αA-R21Q-crystallin at the N-terminal region can rescue a deleterious mutation in the conserved α-crystallin domain of the protein.
Assuntos
Proteínas Recombinantes/metabolismo , Cadeia A de alfa-Cristalina/metabolismo , Álcool Desidrogenase/metabolismo , Sequência de Bases , Linhagem Celular , Sobrevivência Celular/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Insulina/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Multimerização Proteica/genética , Estabilidade Proteica , Desdobramento de Proteína , Proteínas Recombinantes/genética , Supressão Genética , Cadeia A de alfa-Cristalina/genéticaRESUMO
Purpose: The G98R mutant of αA-crystallin is associated with the development of presenile cataracts. In vitro, the recombinant mutant protein exhibits altered structural and functional characteristics, along with the propensity to aggregate by itself and precipitate. Previously, we have reported that the N-terminal aspartate substituted form of the antiaggregation peptide, D71FVIFLDVKHFSPEDLTVK88 (αA-minichaperone or mini-αA) prevented aggregation of αAG98R. However, the mechanism of stabilization of αAG98R from aggregation is not fully understood. The purpose of this study was to determine whether the surface charge (zeta (ζ) potential) of αAG98R in the presence of the peptide chaperone contributed to the stabilization of mutant protein, and to identify the sites of interaction between αAG98R and the peptide chaperone. Methods: Wild-type αA-crystallin (αAWT) and recombinant mutant αAG98R were purified from Escherichia coli BL21(DE3)pLysS cells. The ζ potential values of αA-crystallins in the presence or absence of αA-minichaperone and purified protein-peptide complexes were estimated in a ζ potential analyzer. Potential regions within αAG98R that bind the αA-minichaperone were investigated by incubating the protein with a photoactivable minichaperone variant, followed by mass spectrometric analysis. Results: Binding of the αA-minichaperone to aggregation-prone αAG98R was accompanied by an increase in the ζ potential from -15.19±0.870 mV corresponding to αAG98R alone to -28.64±1.640 mV for the purified complex. Mass spectrometric analysis identified 1MDVTIQHPWFK11, 13TLGPFYPSR21, 55TVLDSGISEVR65, and 113EFHRR117 as the αA-minichaperone-binding regions in αAG98R. The results suggest the involvement of the N-terminal region and the α-crystallin domain in the peptide-mediated stabilization of αAG98R. Conclusions: The αA-crystallin-derived minichaperone stabilizes αAG98R by compensating its lost surface charge. Methods for increasing the ζ potential of aggregating proteins can be a potential approach for therapy to protein aggregation diseases.
Assuntos
Chaperonas Moleculares/química , Mutação , Peptídeos/química , Agregados Proteicos , Cadeia A de alfa-Cristalina/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Cadeia A de alfa-Cristalina/genética , Cadeia A de alfa-Cristalina/metabolismoRESUMO
Several mutations associated with congenital cataracts in human beings target conserved arginine residues in αA-crystallin. The N-terminal region of αA-crystallin is a "mutational hotspot," with multiple cataract-related mutations reported in this region. Two mutations at arginine 21 in the N-terminal domain of αA-crystallin - αA-R21L and αA-R21W have been associated with congenital cataract. A third mutant of R21, αA-R21Q, was recently identified to be associated with congenital cataract in a South Australian family. The point mutation was reported to compromise the quaternary structure of αA-crystallin by preventing its assembly into higher ordered oligomers. To assess the effect of the αA-R21Q mutation on αA-crystallin function, recombinant αA-R21Q was expressed, purified and characterized in vitro. Compared to wild-type αA-crystallin, the recombinant αA-R21Q exhibits enhanced chaperone-like activity, increased surface hydrophobicity, lesser stability in urea and increased susceptibility to digestion by trypsin. αA-R21Q demonstrated increased binding affinity towards unfolding ADH and bovine lens fiber cell membranes. αA-R21Q homo-oligomers and hetero-oligomers also prevented H2O2-induced apoptosis in ARPE-19â¯cells. Taken together, αA-R21Q exhibited a gain of function despite subtle structural differences as compared to wild-type αA-crystallin. This study further validates the involvement of arginine 21 in regulating αA-crystallin structure and function.
Assuntos
Catarata , Cristalino/química , Cadeia A de alfa-Cristalina , Animais , Apoptose/fisiologia , Arginina/genética , Arginina/fisiologia , Catarata/genética , Catarata/metabolismo , Bovinos , Humanos , Chaperonas Moleculares/fisiologia , Mutação Puntual , Ligação Proteica/fisiologia , Dobramento de Proteína , Ureia/metabolismo , Cadeia A de alfa-Cristalina/química , Cadeia A de alfa-Cristalina/genética , Cadeia A de alfa-Cristalina/fisiologiaRESUMO
Expression and secretion of neurotrophic factors have long been known as a key mechanism of neuroglial interaction in the central nervous system. In addition, several other intrinsic neuroprotective pathways have been described, including those involving small heat shock proteins such as α-crystallins. While initially considered as a purely intracellular mechanism, both αA-crystallins and αB-crystallins have been recently reported to be secreted by glial cells. While an anti-apoptotic effect of such secreted αA-crystallin has been suggested, its regulation and protective potential remain unclear. We recently identified residue threonine 148 (T148) and its phosphorylation as a critical regulator of αA-crystallin intrinsic neuroprotective function. In the current study, we explored how mutation of this residue affected αA-crystallin chaperone function, secretion, and paracrine protective function using primary glial and neuronal cells. After demonstrating the paracrine protective effect of αA-crystallins secreted by primary Müller glial cells (MGCs), we purified and characterized recombinant αA-crystallin proteins mutated on the T148 regulatory residue. Characterization of the biochemical properties of these mutants revealed an increased chaperone activity of the phosphomimetic T148D mutant. Consistent with this observation, we also show that exogeneous supplementation of the phosphomimetic T148D mutant protein protected primary retinal neurons from metabolic stress despite similar cellular uptake. In contrast, the nonphosphorylatable mutant was completely ineffective. Altogether, our study demonstrates the paracrine role of αA-crystallin in the central nervous system as well as the therapeutic potential of functionally enhanced αA-crystallin recombinant proteins to prevent metabolic-stress induced neurodegeneration.
Assuntos
Cristalinas , Cristalinas/química , Cristalinas/genética , Cristalinas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
The chaperone and anti-apoptotic activity of α-crystallins (αA- and αB-) and their derivatives has received increasing attention due to their tremendous potential in preventing cell death. While originally known and described for their role in the lens, the upregulation of these proteins in cells and animal models of neurodegenerative diseases highlighted their involvement in adaptive protective responses to neurodegeneration associated stress. However, several studies also suggest that chronic neurodegenerative conditions are associated with progressive loss of function of these proteins. Thus, while external supplementation of α-crystallin shows promise, their potential as a protein-based therapeutic for the treatment of chronic neurodegenerative diseases remains ambiguous. The current review aims at assessing the current literature supporting the anti-apoptotic potential of αA- and αB-crystallins and its potential involvement in retinal neurodegenerative diseases. The review further extends into potentially modulating the chaperone and the anti-apoptotic function of α-crystallins and the use of such functionally enhanced proteins for promoting neuronal viability in retinal neurodegenerative disease.
RESUMO
The paradigm that cataracts are irreversible and that vision from cataracts can only be restored through surgery has recently been challenged by reports that oxysterols such as lanosterol and 25-hydroxycholesterol can restore vision by binding to αB-crystallin chaperone protein to dissolve or disaggregate lenticular opacities. To confirm this premise, in vitro rat lens studies along with human lens protein solubilization studies were conducted. Cataracts were induced in viable rat lenses cultured for 48 hours in TC-199 bicarbonate media through physical trauma, 10 mM ouabain as Na+/K+ ATPase ion transport inhibitor, or 1 mM of an experimental compound that induces water influx into the lens. Subsequent 48-hour incubation with 15 mM of lanosterol liposomes failed to either reverse these lens opacities or prevent the further progression of cataracts to the nuclear stage. Similarly, 3-day incubation of 47-year old human lenses in media containing 0.20 mM lanosterol or 60-year-old human lenses in 0.25 and 0.50 mM 25-hydroxycholesterol failed to increase the levels of soluble lens proteins or decrease the levels of insoluble lens proteins. These binding studies were followed up with in silico binding studies of lanosterol, 25-hydroxycholesterol, and ATP as a control to two wild type (2WJ7 and 2KLR) and one R120G mutant (2Y1Z) αB-crystallins using standard MOETM (Molecular Operating Environment) and Schrödinger's Maestro software. Results confirmed that compared to ATP, both oxysterols failed to reach the acceptable threshold binding scores for good predictive binding to the αB-crystallins. In summary, all three studies failed to provide evidence that lanosterol or 25-hydroxycholesterol have either anti-cataractogenic activity or bind aggregated lens protein to dissolve cataracts.