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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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Disturbed oligomerization of variant MCAD proteins is partially rescued by co-overexpression of GroESL. Oligomerization profiles of wild-type (WT) and variant MCAD proteins were determined by size-exclusion chromatography. Soluble high molecular weight aggregates (A) eluted at volumes of 45–47 ml, tetramers (T) at 58–62 ml. No dimers or monomers were observed with any of the variants. Elution volumes of >85 ml contained fragments (F) of degraded MCAD or MBP. (A) Profiles without co-overexpression of GroESL. Wild-type (WT) and R388S almost exclusively eluted as tetramers. All other variants eluted as high molecular weight aggregates or low molecular weight fragments with only three variants (Y42H, D241G and R309K) showing small peaks of tetrameric MCAD. Aggregates and degradation products of Y133H and R181C comprised various molecular weights spread all over the chromatogram. (B) Profiles with co-overexpression of GroESL. Variants A27V, Y42H, Y133H, R181C, D241G, K304E and R309K showed a rescue of tetramer formation with only small amounts of aggregates. Only for variant I331T, tetramer formation could not be restored. Note: for WT, the chromatogram without co-overexpression of GroESL is depicted.
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DDP079F2: Disturbed oligomerization of variant MCAD proteins is partially rescued by co-overexpression of GroESL. Oligomerization profiles of wild-type (WT) and variant MCAD proteins were determined by size-exclusion chromatography. Soluble high molecular weight aggregates (A) eluted at volumes of 45–47 ml, tetramers (T) at 58–62 ml. No dimers or monomers were observed with any of the variants. Elution volumes of >85 ml contained fragments (F) of degraded MCAD or MBP. (A) Profiles without co-overexpression of GroESL. Wild-type (WT) and R388S almost exclusively eluted as tetramers. All other variants eluted as high molecular weight aggregates or low molecular weight fragments with only three variants (Y42H, D241G and R309K) showing small peaks of tetrameric MCAD. Aggregates and degradation products of Y133H and R181C comprised various molecular weights spread all over the chromatogram. (B) Profiles with co-overexpression of GroESL. Variants A27V, Y42H, Y133H, R181C, D241G, K304E and R309K showed a rescue of tetramer formation with only small amounts of aggregates. Only for variant I331T, tetramer formation could not be restored. Note: for WT, the chromatogram without co-overexpression of GroESL is depicted.

Mentions: Wild-type and ten variant forms of MCAD (Fig. 1 and Table 1) were purified by affinity chromatography and subsequent size-exclusion chromatography (SEC) to analyze the oligomeric states of the expressed fusion proteins (Fig. 2). Wild-type MCAD was eluted in the tetrameric form with an almost negligible amount of aggregates (<1%), whereas MCAD variants showed a markedly decreased expression of soluble protein consistent with proneness to aggregation and degradation of misfolded protein. Only one variant (R388S) showed an elution profile identical to wild-type. The remaining variants revealed severely disturbed oligomerization consisting of (i) small amounts of tetramers (Y42H, D241G, R309K) with or without high molecular weight aggregates, (ii) exclusively high molecular weight aggregates (A27V, K304E, I331T) or (iii) small amounts of protein fragments of various molecular weights (Y133H, R181C) (Fig. 2A). No monomers or dimers were observed with any of the variants. R223G was expressed as truncated, instable protein with no detectable activity (data not shown) and could not be subjected to SEC.


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)

Disturbed oligomerization of variant MCAD proteins is partially rescued by co-overexpression of GroESL. Oligomerization profiles of wild-type (WT) and variant MCAD proteins were determined by size-exclusion chromatography. Soluble high molecular weight aggregates (A) eluted at volumes of 45–47 ml, tetramers (T) at 58–62 ml. No dimers or monomers were observed with any of the variants. Elution volumes of >85 ml contained fragments (F) of degraded MCAD or MBP. (A) Profiles without co-overexpression of GroESL. Wild-type (WT) and R388S almost exclusively eluted as tetramers. All other variants eluted as high molecular weight aggregates or low molecular weight fragments with only three variants (Y42H, D241G and R309K) showing small peaks of tetrameric MCAD. Aggregates and degradation products of Y133H and R181C comprised various molecular weights spread all over the chromatogram. (B) Profiles with co-overexpression of GroESL. Variants A27V, Y42H, Y133H, R181C, D241G, K304E and R309K showed a rescue of tetramer formation with only small amounts of aggregates. Only for variant I331T, tetramer formation could not be restored. Note: for WT, the chromatogram without co-overexpression of GroESL is depicted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2667288&req=5

DDP079F2: Disturbed oligomerization of variant MCAD proteins is partially rescued by co-overexpression of GroESL. Oligomerization profiles of wild-type (WT) and variant MCAD proteins were determined by size-exclusion chromatography. Soluble high molecular weight aggregates (A) eluted at volumes of 45–47 ml, tetramers (T) at 58–62 ml. No dimers or monomers were observed with any of the variants. Elution volumes of >85 ml contained fragments (F) of degraded MCAD or MBP. (A) Profiles without co-overexpression of GroESL. Wild-type (WT) and R388S almost exclusively eluted as tetramers. All other variants eluted as high molecular weight aggregates or low molecular weight fragments with only three variants (Y42H, D241G and R309K) showing small peaks of tetrameric MCAD. Aggregates and degradation products of Y133H and R181C comprised various molecular weights spread all over the chromatogram. (B) Profiles with co-overexpression of GroESL. Variants A27V, Y42H, Y133H, R181C, D241G, K304E and R309K showed a rescue of tetramer formation with only small amounts of aggregates. Only for variant I331T, tetramer formation could not be restored. Note: for WT, the chromatogram without co-overexpression of GroESL is depicted.
Mentions: Wild-type and ten variant forms of MCAD (Fig. 1 and Table 1) were purified by affinity chromatography and subsequent size-exclusion chromatography (SEC) to analyze the oligomeric states of the expressed fusion proteins (Fig. 2). Wild-type MCAD was eluted in the tetrameric form with an almost negligible amount of aggregates (<1%), whereas MCAD variants showed a markedly decreased expression of soluble protein consistent with proneness to aggregation and degradation of misfolded protein. Only one variant (R388S) showed an elution profile identical to wild-type. The remaining variants revealed severely disturbed oligomerization consisting of (i) small amounts of tetramers (Y42H, D241G, R309K) with or without high molecular weight aggregates, (ii) exclusively high molecular weight aggregates (A27V, K304E, I331T) or (iii) small amounts of protein fragments of various molecular weights (Y133H, R181C) (Fig. 2A). No monomers or dimers were observed with any of the variants. R223G was expressed as truncated, instable protein with no detectable activity (data not shown) and could not be subjected to SEC.

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