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Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening.

Maier EM, Gersting SW, Kemter KF, Jank JM, Reindl M, Messing DD, Truger MS, Sommerhoff CP, Muntau AC - Hum. Mol. Genet. (2009)

Bottom Line: This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants.Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity.Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pediatrics, Children's Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT
Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the beta-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.

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Variant MCAD proteins show accelerated thermal denaturation and partial protein unfolding in the ground state for some variants. Thermal unfolding of wild-type (WT) and variants monitored by ANS fluorescence. (A) ANS fluorescence profiles of wild-type, Y133H, R181S and K304E. Intensities of the fluorescent dye ANS, which binds to hydrophobic groups of the protein presented upon unfolding, are plotted as a function of increasing temperatures. Ground-state fluorescence was markedly increased for Y133H and, to a lesser extent, for R181C and K304E indicating an increased hydrophobicity due to partial unfolding of these variants already in the native state. (B) Thermal denaturation of all variants analyzed. Fractions of unfolded protein are plotted as a function of increasing temperatures and the transition midpoints represent the temperature at half denaturation (fraction unfolded 0.5). All variants showed a marked to moderate left-shift of the curves implying an increased propensity to unfold upon thermal stress (Table 5). In addition, Y133H showed accelerated unfolding as indicated by the steeper slope of the curve and complete denaturation at 46°C.
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DDP079F4: Variant MCAD proteins show accelerated thermal denaturation and partial protein unfolding in the ground state for some variants. Thermal unfolding of wild-type (WT) and variants monitored by ANS fluorescence. (A) ANS fluorescence profiles of wild-type, Y133H, R181S and K304E. Intensities of the fluorescent dye ANS, which binds to hydrophobic groups of the protein presented upon unfolding, are plotted as a function of increasing temperatures. Ground-state fluorescence was markedly increased for Y133H and, to a lesser extent, for R181C and K304E indicating an increased hydrophobicity due to partial unfolding of these variants already in the native state. (B) Thermal denaturation of all variants analyzed. Fractions of unfolded protein are plotted as a function of increasing temperatures and the transition midpoints represent the temperature at half denaturation (fraction unfolded 0.5). All variants showed a marked to moderate left-shift of the curves implying an increased propensity to unfold upon thermal stress (Table 5). In addition, Y133H showed accelerated unfolding as indicated by the steeper slope of the curve and complete denaturation at 46°C.

Mentions: Figure 4A shows ANS fluorescence profiles upon thermal denaturation of wild-type, K304E and two severely distorted variants. In variant Y133H, a considerably (20-fold) elevated ground state fluorescence signal at 25°C was detected in comparison with wild-type. This finding indicates an increased hydrophobicity due to partial protein unfolding even without the application of thermal stress. The same was found in R181C and K304E, but to a much lesser extent. Upon thermal denaturation, for all MCAD variants the transition from the native state to the unfolded state occurred at significantly lower temperatures than in the wild-type (Fig. 4B and Table 5). The variants Y133H and R181C showed the most pronounced alterations with Tm1/2 of 42.2 and 40.0°C, respectively. In addition to the marked left-shift of the curve, Y133H revealed a steeper slope of the curve indicating an accelerated progress of unfolding with complete denaturation at 46°C.


Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening.

Maier EM, Gersting SW, Kemter KF, Jank JM, Reindl M, Messing DD, Truger MS, Sommerhoff CP, Muntau AC - Hum. Mol. Genet. (2009)

Variant MCAD proteins show accelerated thermal denaturation and partial protein unfolding in the ground state for some variants. Thermal unfolding of wild-type (WT) and variants monitored by ANS fluorescence. (A) ANS fluorescence profiles of wild-type, Y133H, R181S and K304E. Intensities of the fluorescent dye ANS, which binds to hydrophobic groups of the protein presented upon unfolding, are plotted as a function of increasing temperatures. Ground-state fluorescence was markedly increased for Y133H and, to a lesser extent, for R181C and K304E indicating an increased hydrophobicity due to partial unfolding of these variants already in the native state. (B) Thermal denaturation of all variants analyzed. Fractions of unfolded protein are plotted as a function of increasing temperatures and the transition midpoints represent the temperature at half denaturation (fraction unfolded 0.5). All variants showed a marked to moderate left-shift of the curves implying an increased propensity to unfold upon thermal stress (Table 5). In addition, Y133H showed accelerated unfolding as indicated by the steeper slope of the curve and complete denaturation at 46°C.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667288&req=5

DDP079F4: Variant MCAD proteins show accelerated thermal denaturation and partial protein unfolding in the ground state for some variants. Thermal unfolding of wild-type (WT) and variants monitored by ANS fluorescence. (A) ANS fluorescence profiles of wild-type, Y133H, R181S and K304E. Intensities of the fluorescent dye ANS, which binds to hydrophobic groups of the protein presented upon unfolding, are plotted as a function of increasing temperatures. Ground-state fluorescence was markedly increased for Y133H and, to a lesser extent, for R181C and K304E indicating an increased hydrophobicity due to partial unfolding of these variants already in the native state. (B) Thermal denaturation of all variants analyzed. Fractions of unfolded protein are plotted as a function of increasing temperatures and the transition midpoints represent the temperature at half denaturation (fraction unfolded 0.5). All variants showed a marked to moderate left-shift of the curves implying an increased propensity to unfold upon thermal stress (Table 5). In addition, Y133H showed accelerated unfolding as indicated by the steeper slope of the curve and complete denaturation at 46°C.
Mentions: Figure 4A shows ANS fluorescence profiles upon thermal denaturation of wild-type, K304E and two severely distorted variants. In variant Y133H, a considerably (20-fold) elevated ground state fluorescence signal at 25°C was detected in comparison with wild-type. This finding indicates an increased hydrophobicity due to partial protein unfolding even without the application of thermal stress. The same was found in R181C and K304E, but to a much lesser extent. Upon thermal denaturation, for all MCAD variants the transition from the native state to the unfolded state occurred at significantly lower temperatures than in the wild-type (Fig. 4B and Table 5). The variants Y133H and R181C showed the most pronounced alterations with Tm1/2 of 42.2 and 40.0°C, respectively. In addition to the marked left-shift of the curve, Y133H revealed a steeper slope of the curve indicating an accelerated progress of unfolding with complete denaturation at 46°C.

Bottom Line: This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants.Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity.Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pediatrics, Children's Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT
Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the beta-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.

Show MeSH
Related in: MedlinePlus