Glutaryl-CoA Dehydrogenase Misfolding in Glutaric Acidemia Type 1.

Glutaric acidemia type 1 (GA1) is a neurotoxic metabolic disorder due to glutaryl-CoA dehydrogenase (GCDH) deficiency. The high number of missense variants associated with the disease and their impact on GCDH activity suggest that disturbed protein conformation can affect the biochemical phenotype. We aimed to elucidate the molecular basis of protein loss of function in GA1 by performing a parallel analysis in a large panel of GCDH missense variants using different biochemical and biophysical methodologies. Thirteen GCDH variants were investigated in regard to protein stability, hydrophobicity, oligomerization, aggregation, and activity. An altered oligomerization, loss of protein stability and solubility, as well as an augmented susceptibility to aggregation were observed. GA1 variants led to a loss of enzymatic activity, particularly when present at the N-terminal domain. The reduced cellular activity was associated with loss of tetramerization. Our results also suggest a correlation between variant sequence location and cellular protein stability (p < 0.05), with a more pronounced loss of protein observed with variant proximity to the N-terminus. The broad panel of variant-mediated conformational changes of the GCDH protein supports the classification of GA1 as a protein-misfolding disorder. This work supports research toward new therapeutic strategies that target this molecular disease phenotype.

Reduced Humoral and Cellular Immune Response to Primary COVID-19 mRNA Vaccination in Kidney Transplanted Children Aged 5-11 Years.

The situation of limited data concerning the response to COVID-19 mRNA vaccinations in immunocom-promised children hinders evidence-based recommendations. This prospective observational study investigated humoral and T cell responses after primary BNT162b2 vaccination in secondary immunocompromised and healthy children aged 5-11 years. Participants were categorized as: children after kidney transplantation (KTx, n = 9), proteinuric glomerulonephritis (GN, n = 4) and healthy children (controls, n = 8). Expression of activation-induced markers and cytokine secretion were determined to quantify the T cell response from PBMCs stimulated with peptide pools covering the spike glycoprotein of SARS-CoV-2 Wuhan Hu-1 and Omicron BA.5. Antibodies against SARS-CoV-2 spike receptor-binding domain were quantified in serum. Seroconversion was detected in 56% of KTx patients and in 100% of the GN patients and controls. Titer levels were significantly higher in GN patients and controls than in KTx patients. In Ktx patients, the humoral response increased after a third immunization. No differences in the frequency of antigen-specific CD4+ and CD8+ T cells between all groups were observed. T cells showed a predominant anti-viral capacity in their secreted cytokines; however, this capacity was reduced in KTx patients. This study provides missing evidence concerning the humoral and T cell response in immunocompromised children after COVID-19 vaccination.

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy.

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9-mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

Long-Term Antibody Response to SARS-CoV-2 in Children.

Almost 2 years into the pandemic and with vaccination of children significantly lagging behind adults, long-term pediatric humoral immune responses to SARS-CoV-2 are understudied. The C19.CHILD Hamburg (COVID-19 Child Health Investigation of Latent Disease) Study is a prospective cohort study designed to identify and follow up children and their household contacts infected in the early 2020 first wave of SARS-CoV-2. We screened 6113 children < 18 years by nasopharyngeal swab-PCR in a low-incidence setting after general lockdown, from May 11 to June 30, 2020. A total of 4657 participants underwent antibody testing. Positive tests were followed up by repeated PCR and serological testing of all household contacts over 6 months. In total, the study identified 67 seropositive children (1.44%); the median time after infection at first presentation was 83 days post-symptom onset (PSO). Follow-up of household contacts showed less than 100% seroprevalence in most families, with higher seroprevalence in families with adult index cases compared to pediatric index cases (OR 1.79, P = 0.047). Most importantly, children showed sustained seroconversion up to 9 months PSO, and serum antibody concentrations persistently surpassed adult levels (ratio serum IgG spike children vs. adults 90 days PSO 1.75, P < 0.001; 180 days 1.38, P = 0.01; 270 days 1.54, P = 0.001). In a low-incidence setting, SARS-CoV-2 infection and humoral immune response present distinct patterns in children including higher antibody levels, and lower seroprevalence in families with pediatric index cases. Children show long-term SARS-CoV-2 antibody responses. These findings are relevant to novel variants with increased disease burden in children, as well as for the planning of age-appropriate vaccination strategies.

Specific CD4+ T Cell Responses to Ancestral SARS-CoV-2 in Children Increase With Age and Show Cross-Reactivity to Beta Variant.

SARS-CoV-2 is still a major burden for global health despite effective vaccines. With the reduction of social distancing measures, infection rates are increasing in children, while data on the pediatric immune response to SARS-CoV-2 infection is still lacking. Although the typical disease course in children has been mild, emerging variants may present new challenges in this age group. Peripheral blood mononuclear cells (PBMC) from 51 convalescent children, 24 seronegative siblings from early 2020, and 51 unexposed controls were stimulated with SARS-CoV-2-derived peptide MegaPools from the ancestral and beta variants. Flow cytometric determination of activation-induced markers and secreted cytokines were used to quantify the CD4+ T cell response. The average time after infection was over 80 days. CD4+ T cell responses were detected in 61% of convalescent children and were markedly reduced in preschool children. Cross-reactive T cells for the SARS-CoV-2 beta variant were identified in 45% of cases after infection with an ancestral SARS-CoV-2 variant. The CD4+ T cell response was accompanied most predominantly by IFN-γ and Granzyme B secretion. An antiviral CD4+ T cell response was present in children after ancestral SARS-CoV-2 infection, which was reduced in the youngest age group. We detected significant cross-reactivity of CD4+ T cell responses to the more recently evolved immune-escaping beta variant. Our findings have epidemiologic relevance for children regarding novel viral variants of concern and vaccination efforts.

Phenotypic analysis of the pediatric immune response to SARS-CoV-2 by flow cytometry.

Pediatric SARS-CoV-2 infection is often mild or asymptomatic and the immune responses of children are understudied compared to adults. Here, we present and evaluate the performance of a two-panel (16- and 17 parameter) flow cytometry-based approach for immune phenotypic analysis of cryopreserved PBMC samples from children after SARS-CoV-2 infection. The panels were optimized based on previous SARS-CoV-2 related studies for the pediatric immune system. PBMC samples from seven SARS-CoV-2 seropositive children from early 2020 and five age-matched healthy controls were stained for analysis of T-cells (panel T), B and innate immune cells (panel B). Performance of the panels was evaluated in two parallel approaches, namely classical manual gating of known subpopulations and unbiased clustering using the R-based algorithm PhenoGraph. Using manual gating we clearly identified 14 predefined subpopulations of interest for panel T and 19 populations in panel B in low-volume pediatric samples. PhenoGraph found 18 clusters within the T-cell panel and 21 clusters within the innate and B-cell panel that could be unmistakably annotated. Combining the data of the two panels and analysis approaches, we found expected differentially abundant clusters in SARS-CoV-2 seropositive children compared to healthy controls, underscoring the value of these two panels for the analysis of immune response to SARS-CoV-2. We established a two-panel flow cytometry approach that can be used with limited amounts of cryopreserved pediatric samples. Our workflow allowed for a rapid, comprehensive, and robust pediatric immune phenotyping with comparable performance in manual gating and unbiased clustering. These panels may be adapted for large multi-center cohort studies to investigate the pediatric immune response to emerging virus variants in the ongoing and future pandemics.

Edgetic Perturbations Contribute to Phenotypic Variability in PEX26 Deficiency.

Peroxisomes share metabolic pathways with other organelles and peroxisomes are embedded into key cellular processes. However, the specific function of many peroxisomal proteins remains unclear and restricted knowledge of the peroxisomal protein interaction network limits a precise mapping of this network into the cellular metabolism. Inborn peroxisomal disorders are autosomal or X-linked recessive diseases that affect peroxisomal biogenesis (PBD) and/or peroxisomal metabolism. Pathogenic variants in the PEX26 gene lead to peroxisomal disorders of the full Zellweger spectrum continuum. To investigate the phenotypic complexity of PEX26 deficiency, we performed a combined organelle protein interaction screen and network medicine approach and 1) analyzed whether PEX26 establishes interactions with other peroxisomal proteins, 2) deciphered the PEX26 interaction network, 3) determined how PEX26 is involved in further processes of peroxisomal biogenesis and metabolism, and 4) showed how variant-specific disruption of protein-protein interactions (edgetic perturbations) may contribute to phenotypic variability in PEX26 deficient patients. The discovery of 14 novel protein-protein interactions for PEX26 revealed a hub position of PEX26 inside the peroxisomal interactome. Analysis of edgetic perturbations of PEX26 variants revealed a strong correlation between the number of affected protein-protein interactions and the molecular phenotype of matrix protein import. The role of PEX26 in peroxisomal biogenesis was expanded encompassing matrix protein import, division and proliferation, and membrane assembly. Moreover, the PEX26 interaction network intersects with cellular lipid metabolism at different steps. The results of this study expand the knowledge about the function of PEX26 and refine genotype-phenotype correlations, which may contribute to our understanding of the underlying disease mechanism of PEX26 deficiency.

iBRET Screen of the ABCD1 Peroxisomal Network and Mutation-Induced Network Perturbations.

Mapping the network of proteins provides a powerful means to investigate the function of disease genes and to unravel the molecular basis of phenotypes. We present an automated informatics-aided and bioluminescence resonance energy transfer-based approach (iBRET) enabling high-confidence detection of protein-protein interactions in living mammalian cells. A screen of the ABCD1 protein, which is affected in X-linked adrenoleukodystrophy (X-ALD), against an organelle library of peroxisomal proteins demonstrated applicability of iBRET for large-scale experiments. We identified novel protein-protein interactions for ABCD1 (with ALDH3A2, DAO, ECI2, FAR1, PEX10, PEX13, PEX5, PXMP2, and PIPOX), mapped its position within the peroxisomal protein-protein interaction network, and determined that pathogenic missense variants in ABCD1 alter the interaction with selected binding partners. These findings provide mechanistic insights into pathophysiology of X-ALD and may foster the identification of new disease modifiers.

Multiplexed complexome profiling using tandem mass tags.

Complexome profiling is a rapidly spreading, powerful technique to gain insight into the nature of protein complexes. It identifies and quantifies protein complexes separated into multiple fractions of increasing molecular mass using mass spectrometry-based, label-free bottom-up proteomics. Complexome profiling enables a sophisticated and thorough characterization of the composition, molecular mass, assembly, and interactions of protein complexes. However, in practice, its application is limited by the large number of samples it generates and the related time of mass spectrometry analyses. Here, we report an improved process workflow that implements tandem mass tags for multiplexing complexome profiling. This workflow substantially reduces the number of samples and measuring time without compromising protein identification or quantification reliability. In profiles from mitochondrial fractions of cells recovering from chloramphenicol treatment, tandem mass tags-multiplexed complexome profiling exhibited migration patterns of mature ATP synthase (complex V) and assembly intermediates that were consistent in composition and abundance with profiles obtained by the label-free approach. Reporter ion quantifications of proteins and complexes unaffected by the chloramphenicol treatment presented less variation in comparison to the label-free method. Incorporation of tandem mass tags enabled an efficient and robust complexome profiling analysis and may foster broader application for protein complex profiling in biomedical research and diagnostics.

Isoform-specific domain organization determines conformation and function of the peroxisomal biogenesis factor PEX26.

Peroxisomal biogenesis factor PEX26 is a membrane anchor for the multi-subunit PEX1-PEX6 protein complex that controls ubiquitination and dislocation of PEX5 cargo receptors for peroxisomal matrix protein import. PEX26 associates with the peroxisomal translocation pore via PEX14 and a splice variant (PEX26Δex5) of unknown function has been reported. Here, we demonstrate PEX26 homooligomerization mediated by two heptad repeat domains adjacent to the transmembrane domain. We show that isoform-specific domain organization determines PEX26 oligomerization and impacts peroxisomal ß-oxidation and proliferation. PEX26 and PEX26Δex5 displayed different patterns of interaction with PEX2-PEX10 or PEX13-PEX14 complexes, which relate to distinct pre-peroxisomes in the de novo synthesis pathway. Our data support an alternative PEX14-dependent mechanism of peroxisomal membrane association for the splice variant, which lacks a transmembrane domain. Structure-function relationships of PEX26 isoforms explain an extended function in peroxisomal homeostasis and these findings may improve our understanding of the broad phenotype of PEX26-associated human disorders.

Secondary BH4 deficiency links protein homeostasis to regulation of phenylalanine metabolism.

Metabolic control of phenylalanine concentrations in body fluids is essential for cognitive development and executive function. The hepatic phenylalanine hydroxylating system is regulated by the ratio of l-phenylalanine, which is substrate of phenylalanine hydroxylase (PAH), to the PAH cofactor tetrahydrobiopterin (BH4). Physiologically, phenylalanine availability is governed by nutrient intake, whereas liver BH4 is kept at constant level. In phenylketonuria, PAH deficiency leads to elevated blood phenylalanine and is often caused by PAH protein misfolding with loss of function. Here, we report secondary hepatic BH4 deficiency in Pah-deficient mice. Alterations in de novo synthesis and turnover of BH4 were ruled out as molecular causes. We demonstrate that kinetically instable and aggregation-prone variant Pah proteins trap BH4, shifting the pool of free BH4 towards bound BH4. Interference of PAH protein misfolding with metabolite-based control of l-phenylalanine turnover suggests a mechanistic link between perturbation of protein homeostasis and disturbed regulation of metabolic pathways.

Inborn errors of metabolism and the human interactome: a systems medicine approach.

The group of inborn errors of metabolism (IEM) displays a marked heterogeneity and IEM can affect virtually all functions and organs of the human organism; however, IEM share that their associated proteins function in metabolism. Most proteins carry out cellular functions by interacting with other proteins, and thus are organized in biological networks. Therefore, diseases are rarely the consequence of single gene mutations but of the perturbations caused in the related cellular network. Systematic approaches that integrate multi-omics and database information into biological networks have successfully expanded our knowledge of complex disorders but network-based strategies have been rarely applied to study IEM. We analyzed IEM on a proteome scale and found that IEM-associated proteins are organized as a network of linked modules within the human interactome of protein interactions, the IEM interactome. Certain IEM disease groups formed self-contained disease modules, which were highly interlinked. On the other hand, we observed disease modules consisting of proteins from many different disease groups in the IEM interactome. Moreover, we explored the overlap between IEM and non-IEM disease genes and applied network medicine approaches to investigate shared biological pathways, clinical signs and symptoms, and links to drug targets. The provided resources may help to elucidate the molecular mechanisms underlying new IEM, to uncover the significance of disease-associated mutations, to identify new biomarkers, and to develop novel therapeutic strategies.

Disease-causing mutations affecting surface residues of mitochondrial glutaryl-CoA dehydrogenase impair stability, heteromeric complex formation and mitochondria architecture.

The neurometabolic disorder glutaric aciduria type 1 (GA1) is caused by mutations in the GCDH gene encoding the mitochondrial matrix protein glutaryl-CoA dehydrogenase (GCDH), which forms homo- and heteromeric complexes. Twenty percent of all pathogenic mutations affect single amino acid residues on the surface of GCDH resulting in a severe clinical phenotype. We report here on heterologous expression studies of 18 missense mutations identified in GA1 patients affecting surface amino acids. Western blot and pulse chase experiments revealed that the stability of half of the GCDH mutants was significantly reduced. In silico analyses showed that none of the mutations impaired the 3D structure of GCDH. Immunofluorescence co-localisation studies in HeLa cells demonstrated that all GCDH mutants were correctly translocated into mitochondria. Surprisingly, the expression of p.Arg88Cys GCDH as well as further substitutions by alanine, lysine, or methionine but not histidine or leucine resulted in the disruption of mitochondrial architecture forming longitudinal structures composed of stacks of cristae and partial loss of the outer mitochondrial membrane. The expression of mitochondrial fusion or fission proteins was not affected in these cells. Bioluminescence resonance energy transfer analyses revealed that all GCDH mutants exhibit an increased binding affinity to electron transfer flavoprotein beta, whereas only p.Tyr155His GCDH showed a reduced interaction with dihydrolipoamide succinyl transferase. Our data underscore the impact of GCDH protein interactions mediated by amino acid residues on the surface of GCDH required for proper enzymatic activity.

Homooligomerization of ABCA3 and its functional significance.

ABCA3 is a surfactant lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ATP-binding cassette, sub-family A (ABC1), member 3 (ABCA3) gene cause respiratory distress syndrome in newborns, and chronic interstitial lung disease in children and adults. ABCA3 belongs to the class of full ABC transporters, which are supposed to be functional in their monomeric forms. Although other family members e.g., ABCA1 and ABCC7 have been shown to function as oligomers, the oligomerization state of ABCA3 is unknown. In the present study, the oligomerization of ABCA3 was investigated in cell lysates and crude membrane preparations from transiently and stably transfected 293 cells using blue native PAGE (BN-PAGE), gel filtration and co-immunoprecipitation. Additionally, homooligomerization was examined in vivo in cells using bioluminescence resonance energy transfer (BRET). Using BN-PAGE and gel filtration, we demonstrate that non-denatured ABCA3 exists in different oligomeric forms, with monomers (45%) and tetramers (30%) being the most abundant forms. Furthermore, we also show the existence of 20% dimers and 5% trimers. BRET analyses verified intermolecular interactions in vivo. Our results also demonstrated that the arrest of ABCA3 in the endoplasmic reticulum (ER), either through drug treatment or induced by mutations in ABCA3, inhibited the propensity of the protein to form dimers. Based on our results, we suggest that transporter oligomerization is crucial for ABCA3 function and that a disruption of oligomerization due to mutations represents a novel pathomechanism in ABCA3-associated lung disease.

Quantification of mevalonate-5-phosphate using UPLC-MS/MS for determination of mevalonate kinase activity.

OBJECTIVES: Mevalonate kinase deficiency, a rare autosomal recessive autoinflammatory disease, is caused by mutations in the MVK gene encoding mevalonate kinase (MK). MK catalyzes the phosphorylation of mevalonic acid to mevalonate-5-phosphate (MVAP) in the pathway of isoprenoid and sterol synthesis. The disease phenotype correlates with residual activity ranging from <0.5% for mevalonic aciduria to 1-7% for the milder hyperimmunoglobulinemia D and periodic fever syndrome (HIDS). Hence, assessment of loss-of-function requires high accuracy measurements. We describe a method using isotope dilution UPLC-MS/MS for precise and sensitive determination of MK activity. DESIGN AND METHODS: Wild-type MK and the variant V261A, which is associated with HIDS, were recombinantly expressed in Escherichia coli. Enzyme activity was determined by formation of MVAP over time quantified by isotope dilution UPLC-MS/MS. The method was validated according to the FDA Guidance for Bioanalytical Method Validation. RESULTS: Sensitivity for detection of MAVP by UPLC-MS/MS was improved by derivatization with butanol-HCl (LLOQ, 5.0 fmol) and the method was linear from 0.5 to 250 µmol/L (R(2) > 0.99) with a precision of ≥ 89% and an accuracy of ± 2.7%. The imprecision of the activity assay, including the enzymatic reaction and the UPLC-MS/MS quantification, was 8.3%. The variant V261A showed a significantly decreased activity of 53.1%. CONCLUSION: Accurate determination of MK activity was enabled by sensitive and reproducible detection of MVAP using UPLC-MS/MS. The novel method may improve molecular characterization of MVK mutations, provide robust genotype-phenotype correlations, and accelerate compound screening for drug candidates restoring variant MK activity.

IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction.

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

CLPB mutations cause 3-methylglutaconic aciduria, progressive brain atrophy, intellectual disability, congenital neutropenia, cataracts, movement disorder.

We studied a group of individuals with elevated urinary excretion of 3-methylglutaconic acid, neutropenia that can develop into leukemia, a neurological phenotype ranging from nonprogressive intellectual disability to a prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, and early death. Exome sequencing of two unrelated individuals and subsequent Sanger sequencing of 16 individuals with an overlapping phenotype identified a total of 14 rare, predicted deleterious alleles in CLPB in 14 individuals from 9 unrelated families. CLPB encodes caseinolytic peptidase B homolog ClpB, a member of the AAA+ protein family. To evaluate the relevance of CLPB in the pathogenesis of this syndrome, we developed a zebrafish model and an in vitro assay to measure ATPase activity. Suppression of clpb in zebrafish embryos induced a central nervous system phenotype that was consistent with cerebellar and cerebral atrophy that could be rescued by wild-type, but not mutant, human CLPB mRNA. Consistent with these data, the loss-of-function effect of one of the identified variants (c.1222A>G [p.Arg408Gly]) was supported further by in vitro evidence with the mutant peptides abolishing ATPase function. Additionally, we show that CLPB interacts biochemically with ATP2A2, known to be involved in apoptotic processes in severe congenital neutropenia (SCN) 3 (Kostmann disease [caused by HAX1 mutations]). Taken together, mutations in CLPB define a syndrome with intellectual disability, congenital neutropenia, progressive brain atrophy, movement disorder, cataracts, and 3-methylglutaconic aciduria.

Mapping the functional landscape of frequent phenylalanine hydroxylase (PAH) genotypes promotes personalised medicine in phenylketonuria.

BACKGROUND: In phenylketonuria, genetic heterogeneity, frequent compound heterozygosity, and the lack of functional data for phenylalanine hydroxylase genotypes hamper reliable phenotype prediction and individualised treatment. METHODS: A literature search revealed 690 different phenylalanine hydroxylase genotypes in 3066 phenylketonuria patients from Europe and the Middle East. We determined phenylalanine hydroxylase function of 30 frequent homozygous and compound heterozygous genotypes covering 55% of the study population, generated activity landscapes, and assessed the phenylalanine hydroxylase working range in the metabolic (phenylalanine) and therapeutic (tetrahydrobiopterin) space. RESULTS: Shared patterns in genotype-specific functional landscapes were linked to biochemical and pharmacological phenotypes, where (1) residual activity below 3.5% was associated with classical phenylketonuria unresponsive to pharmacological treatment; (2) lack of defined peak activity induced loss of response to tetrahydrobiopterin; (3) a higher cofactor need was linked to inconsistent clinical phenotypes and low rates of tetrahydrobiopterin response; and (4) residual activity above 5%, a defined peak of activity, and a normal cofactor need were associated with pharmacologically treatable mild phenotypes. In addition, we provide a web application for retrieving country-specific information on genotypes and genotype-specific phenylalanine hydroxylase function that warrants continuous extension, updates, and research on demand. CONCLUSIONS: The combination of genotype-specific functional analyses with biochemical, clinical, and therapeutic data of individual patients may serve as a powerful tool to enable phenotype prediction and to establish personalised medicine strategies for dietary regimens and pharmacological treatment in phenylketonuria.

The domain-specific and temperature-dependent protein misfolding phenotype of variant medium-chain acyl-CoA dehydrogenase.

The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a molecular loss-of-function phenotype. Here we established a comprehensive experimental setup to analyze the structural consequences of eight ACADM missense mutations (p.Ala52Val, p.Tyr67His, p.Tyr158His, p.Arg206Cys, p.Asp266Gly, p.Lys329Glu, p.Arg334Lys, p.Arg413Ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. With fever being the crucial risk factor for metabolic decompensation of patients with MCADD, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. Based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in MCADD depends on the structural region that is affected by missense-induced conformational changes with the central ß-domain being particularly prone to structural derangement and destabilization. Since systematic classification of conformational derangements induced by ACADM mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p.Lys329Glu (K304E), the classical severe mutation, and p.Tyr67His (Y42H), discussed to be mild. Experiments assessing the impact of thermal stress revealed that mutations in the ACADM gene lower the temperature threshold at which MCAD loss-of-function occurs. Consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the MCAD enzyme explaining the life-threatening clinical courses observed during fever episodes. Early and aggressive antipyretic treatment thus may be life-saving in patients suffering from MCADD.

Innovative strategies to treat protein misfolding in inborn errors of metabolism: pharmacological chaperones and proteostasis regulators.

To attain functionality, proteins must fold into their three-dimensional native state. The intracellular balance between protein synthesis, folding, and degradation is constantly challenged by genetic or environmental stress factors. In the last ten years, protein misfolding induced by missense mutations was demonstrated to be the seminal molecular mechanism in a constantly growing number of inborn errors of metabolism. In these cases, loss of protein function results from early degradation of missense-induced misfolded proteins. Increasing knowledge on the proteostasis network and the protein quality control system with distinct mechanisms in different compartments of the cell paved the way for the development of new treatment strategies for conformational diseases using small molecules. These comprise proteostasis regulators that enhance the capacity of the proteostasis network and pharmacological chaperones that specifically bind and rescue misfolded proteins by conformational stabilization. They can be used either alone or in combination, the latter to exploit synergistic effects. Many of these small molecule compounds currently undergo preclinical and clinical pharmaceutical development and two have been approved: saproterin dihydrochloride for the treatment of phenylketonuria and tafamidis for the treatment of transthyretin-related hereditary amyloidosis. Different technologies are exploited for the discovery of new small molecule compounds that belong to the still young class of pharmaceutical products discussed here. These compounds may in the near future improve existing treatment strategies or even offer a first-time treatment to patients suffering from nowadays-untreatable inborn errors of metabolism.