Functional Characterization of a Spectrum of Genetic Variants in a Family with Succinic Semialdehyde Dehydrogenase Deficiency.

Succinic semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme involved in the catabolism of the neurotransmitter γ-amino butyric acid. Pathogenic variants in the gene encoding this enzyme cause SSADH deficiency, a developmental disease that manifests as hypotonia, autism, and epilepsy. SSADH deficiency patients usually have family-specific gene variants. Here, we describe a family exhibiting four different SSADH variants: Val90Ala, Cys93Phe, and His180Tyr/Asn255Asp (a double variant). We provide a structural and functional characterization of these variants and show that Cys93Phe and Asn255Asp are pathogenic variants that affect the stability of the SSADH protein. Due to the impairment of the cofactor NAD+ binding, these variants show a highly reduced enzyme activity. However, Val90Ala and His180Tyr exhibit normal activity and expression. The His180Tyr/Asn255Asp variant exhibits a highly reduced activity as a recombinant species, is inactive, and shows a very low expression in eukaryotic cells. A treatment with substances that support protein folding by either increasing chaperone protein expression or by chemical means did not increase the expression of the pathogenic variants of the SSADH deficiency patient. However, stabilization of the folding of pathogenic SSADH variants by other substances may provide a treatment option for this disease.

Phenotypic correlates of structural and functional protein impairments resultant from ALDH5A1 variants.

To investigate the genotype-to-protein-to-phenotype correlations of succinic semialdehyde dehydrogenase deficiency (SSADHD), an inherited metabolic disorder of γ-aminobutyric acid catabolism. Bioinformatics and in silico mutagenesis analyses of ALDH5A1 variants were performed to evaluate their impact on protein stability, active site and co-factor binding domains, splicing, and homotetramer formation. Protein abnormalities were then correlated with a validated disease-specific clinical severity score and neurological, neuropsychological, biochemical, neuroimaging, and neurophysiological metrics. A total of 58 individuals (1:1 male/female ratio) were affected by 32 ALDH5A1 pathogenic variants, eight of which were novel. Compared to individuals with single homotetrameric or multiple homo and heterotetrameric proteins, those predicted not to synthesize any functional enzyme protein had significantly lower expression of ALDH5A1 (p = 0.001), worse overall clinical outcomes (p = 0.008) and specifically more severe cognitive deficits (p = 0.01), epilepsy (p = 0.04) and psychiatric morbidity (p = 0.04). Compared to individuals with predictions of having no protein or a protein impaired in catalytic functions, subjects whose proteins were predicted to be impaired in stability, folding, or oligomerization had a better overall clinical outcome (p = 0.02) and adaptive skills (p = 0.04). The quantity and type of enzyme proteins (no protein, single homotetramers, or multiple homo and heterotetramers), as well as their structural and functional impairments (catalytic or stability, folding, or oligomerization), contribute to phenotype severity in SSADHD. These findings are valuable for assessment of disease prognosis and management, including patient selection for gene replacement therapy. Furthermore, they provide a roadmap to determine genotype-to-protein-to-phenotype relationships in other autosomal recessive disorders.

Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up New Ways of Inhibiting HIV-1 Fusion.

Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env) plays a central role by promoting membrane fusion. We previously identified a vulnerability at the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein increased its inhibitory activity, without altering the binding affinity. Here, to further study the effect of conformational stability on binding and inhibitory potency, we additionally stabilized these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41 CHR region.

Phenotypic Correlates of Structural and Functional Protein Impairments Resultant from ALDH5A1 Variants.

Objective: To investigate the genotype-to-protein-to-phenotype correlations of succinic semialdehyde dehydrogenase deficiency (SSADHD), an inherited metabolic disorder of γ-aminobutyric acid catabolism. Methods: Bioinformatics and in silico mutagenesis analyses of ALDH5A1 variants were performed to evaluate their impact on protein stability, active site and co-factor binding domains, splicing, and homotetramer formation. Protein abnormalities were then correlated with a validated disease-specific clinical severity score and neurological, neuropsychological, biochemical, neuroimaging, and neurophysiological metrics. Results: A total of 58 individuals (1:1 male/female ratio) were affected by 32 ALDH5A1 pathogenic variants, eight of which were novel. Compared to individuals with single homotetrameric or multiple homo and heterotetrameric proteins, those predicted not to synthesize any functional enzyme protein had significantly lower expression of ALDH5A1 (p = 0.001), worse overall clinical outcomes (p = 0.008) and specifically more severe cognitive deficits (p = 0.01), epilepsy (p = 0.04) and psychiatric morbidity (p = 0.04). Compared to individuals with predictions of having no protein or a protein impaired in catalytic functions, subjects whose proteins were predicted to be impaired in stability, folding, or oligomerization had a better overall clinical outcome (p = 0.02) and adaptive skills (p = 0.04). Conclusions: The quantity and type of enzyme proteins (no protein, single homotetramers, or multiple homo and heterotetramers), as well as their structural and functional impairments (catalytic or stability, folding, or oligomerization), contribute to phenotype severity in SSADHD. These findings are valuable for assessment of disease prognosis and management, including patient selection for gene replacement therapy. Furthermore, they provide a roadmap to determine genotype-to-protein-to-phenotype relationships in other autosomal recessive disorders.

Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity.

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR-CHR binding.

Insight into the molecular mechanism underlying the inhibition of α-synuclein aggregation by hydroxytyrosol.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease in the elderly people. To date, drugs able to reverse the disease are not available; the gold standard is levodopa that only relieves clinical symptoms, yet with severe side effects after prolonged administration. Many efforts are underway to find alternative targets for PD prevention or treatment, the most promising being α-synuclein (Syn). Recently, we reported that oleuropein aglycone (OleA) interferes with amyloid aggregation of Syn both stabilizing its monomeric state and inducing the formation of harmless, off-pathway oligomers. This study is focused at describing the interaction between Syn and hydroxytyrosol (HT), the phenolic moiety and main metabolite of OleA, and the interferences with Syn aggregation by using biophysical and biological techniques. Our results show that HT dose-dependently inhibits Syn aggregation and that covalent and non-covalent binding mediate HT-Syn interaction. HT does not modify the natively unfolded structure of Syn, rather, it stabilizes specific regions of the molecule leading to inhibition of protein fibrillation. Cellular assays showed that HT reduces the toxicity of Syn aggregates. Moreover, Syn aggregates interaction with the cell membrane, an important factor for prion-like properties of Syn on-pathway oligomers, was reduced in cells exposed to Syn aggregates grown in the presence of HT.