Galactose epimerase deficiency: lessons from the GalNet registry.

BACKGROUND: Galactose epimerase (GALE) deficiency is a rare hereditary disorder of galactose metabolism with only a few cases described in the literature. This study aims to present the data of patients with GALE deficiency from different countries included through the Galactosemia Network to further expand the existing knowledge and review the current diagnostic strategy, treatment and follow-up of this not well characterized entity. METHODS: Observational study collecting medical data from December 2014 to April 2022 of 22 not previously reported patients from 14 centers in 9 countries. Patients were classified as generalized or non-generalized based on their genotype, enzyme activities in different tissues and/or clinical picture and professional judgment of the treating physician. RESULTS: In total 6 patients were classified as generalized and 16 as non-generalized. In the generalized group, acute neonatal illness was reported in 3, cognitive and developmental delays were present in 5 and hearing problems were reported in 3. Four generalized patients were homozygous for the genetic variant NM_001008216.2:c.280G > A (p.Val94Met). In the non-generalized group, no clearly related symptoms were found. Ten novel genetic variants were reported in this study population. CONCLUSION: The phenotypic spectrum of GALE deficiency ranges from asymptomatic to severe. The generalized patients have a phenotype that is in line with the 9 described cases in the literature and prescribing dietary interventions is the cornerstone for treatment. In the non-generalized group, treatment advice is more difficult. To be able to offer proper counseling, in addition to red blood cell enzyme activity, genetic studies, transferrin glycoform analysis and enzymatic measurements in fibroblasts are recommended. Due to lack of facilities, additional enzymatic testing is not common practice in many centers nor a tailored long-term follow-up is performed.

Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1: Implications of Genetic Diversity of NQO1.

HIF-1α is a master regulator of oxygen homeostasis involved in different stages of cancer development. Thus, HIF-1α inhibition represents an interesting target for anti-cancer therapy. It was recently shown that the HIF-1α interaction with NQO1 inhibits proteasomal degradation of the former, thus suggesting that targeting the stability and/or function of NQO1 could lead to the destabilization of HIF-1α as a therapeutic approach. Since the molecular interactions of NQO1 with HIF-1α are beginning to be unraveled, in this review we discuss: (1) Structure-function relationships of HIF-1α; (2) our current knowledge on the intracellular functions and stability of NQO1; (3) the pharmacological modulation of NQO1 by small ligands regarding function and stability; (4) the potential effects of genetic variability of NQO1 in HIF-1α levels and function; (5) the molecular determinants of NQO1 as a chaperone of many different proteins including cancer-associated factors such as HIF-1α, p53 and p73α. This knowledge is then further discussed in the context of potentially targeting the intracellular stability of HIF-1α by acting on its chaperone, NQO1. This could result in novel anti-cancer therapies, always considering that the substantial genetic variability in NQO1 would likely result in different phenotypic responses among individuals.

Expression, purification and crystallization of a novel metagenome-derived salicylaldehyde dehydrogenase from Alpine soil.

Salicylaldehyde dehydrogenase (SALD) catalyses the last reaction in the upper pathway of naphthalene degradation: the oxidation of salicylaldehyde to salicylate. This enzyme has been isolated and studied from a few organisms that belong to the betaproteobacteria and gammaproteobacteria, predominantly Pseudomonas putida. Furthermore, there is only one crystal structure of this enzyme, which was obtained from P. putida G7. Here, crystallographic studies and analysis of the crystal structure of an Alpine soil metagenome-derived SALD (SALDAP) from an alphaproteobacterium are presented. The SALDAP gene was discovered using gene-targeted sequence assembly and it was cloned into a pLATE51 vector. The recombinant protein was overexpressed in Escherichia coli BL21 (DE3) cells and the soluble protein was purified to homogeneity. The protein crystallized at 20°C and diffraction data from the crystals were collected at a resolution of 1.9 Å. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 116.8, b = 121.7, c = 318.0 Å. Analysis of the crystal structure revealed its conformation to be similar to the organization of the aldehyde dehydrogenase superfamily with three domains: the catalytic, NAD+-binding and bridging domains. The crystal structure of NahF from P. putida G7 was found to be the best structural homologue of SALDAP, even though the enzymes share only 48% amino-acid identity. Interestingly, a carboxylic acid (protocatechuic acid) was found to be a putative ligand of the enzyme and differential scanning fluorimetry was employed to confirm ligand binding. These findings open up the possibility of studying the mechanism(s) of product inhibition and biocatalysis of carboxylic acids using this enzyme and other related aldehyde dehydrogenases.

[13C]-galactose breath test in a patient with galactokinase deficiency and spastic diparesis.

Galactokinase deficiency is an inborn error of carbohydrate metabolism due to a block in the formation of galactose-1-phosphate from galactose. Although the association of galactokinase deficiency with formation of cataracts is well established, the extent of the clinical phenotype is still under investigation. We describe a 6-year-old female who was diagnosed with galactokinase deficiency due to cataract formation when she was 10 months of age and initially started on galactose-restricted diet at that time for 5 months. She developed gait abnormality at 4 years of age. Breath tests via measurement of 13C isotope in exhaled carbon dioxide following 13C-labeled galactose administration at carbon-1 and carbon-2 positions revealed oxidation rates within the normal range. The results in this patient strikingly contrast with the results of another patient with GALK1 deficiency that underwent breath testing with [1-14C]-galactose and [2-14C]-galactose. Extension of in vivo breath tests to other galactokinase patients is needed to better understand the pathophysiology of this disease.

Galactosemia: Towards Pharmacological Chaperones.

Galactosemia is a rare inherited metabolic disease resulting from mutations in the four genes which encode enzymes involved in the metabolism of galactose. The current therapy, the removal of galactose from the diet, is inadequate. Consequently, many patients suffer lifelong physical and cognitive disability. The phenotype varies from almost asymptomatic to life-threatening disability. The fundamental biochemical cause of the disease is a decrease in enzymatic activity due to failure of the affected protein to fold and/or function correctly. Many novel therapies have been proposed for the treatment of galactosemia. Often, these are designed to treat the symptoms and not the fundamental cause. Pharmacological chaperones (PC) (small molecules which correct the folding of misfolded proteins) represent an exciting potential therapy for galactosemia. In theory, they would restore enzyme function, thus preventing downstream pathological consequences. In practice, no PCs have been identified for potential application in galactosemia. Here, we review the biochemical basis of the disease, identify opportunities for the application of PCs and describe how these might be discovered. We will conclude by considering some of the clinical issues which will affect the future use of PCs in the treatment of galactosemia.

Galactokinase deficiency: lessons from the GalNet registry.

PURPOSE: Galactokinase (GALK1) deficiency is a rare hereditary galactose metabolism disorder. Beyond cataract, the phenotypic spectrum is questionable. Data from affected patients included in the Galactosemias Network registry were collected to better characterize the phenotype. METHODS: Observational study collecting medical data of 53 not previously reported GALK1 deficient patients from 17 centers in 11 countries from December 2014 to April 2020. RESULTS: Neonatal or childhood cataract was reported in 15 and 4 patients respectively. The occurrence of neonatal hypoglycemia and infection were comparable with the general population, whereas bleeding diathesis (8.1% versus 2.17-5.9%) and encephalopathy (3.9% versus 0.3%) were reported more often. Elevated transaminases were seen in 25.5%. Cognitive delay was reported in 5 patients. Urinary galactitol was elevated in all patients at diagnosis; five showed unexpected Gal-1-P increase. Most patients showed enzyme activities ≤1%. Eleven different genotypes were described, including six unpublished variants. The majority was homozygous for NM_000154.1:c.82C>A (p.Pro28Thr). Thirty-five patients were diagnosed following newborn screening, which was clearly beneficial. CONCLUSION: The phenotype of GALK1 deficiency may include neonatal elevation of transaminases, bleeding diathesis, and encephalopathy in addition to cataract. Potential complications beyond the neonatal period are not systematically surveyed and a better delineation is needed.

The structural and molecular biology of type IV galactosemia.

Type IV galactosemia is a recently discovered inherited metabolic disease. It is caused by mutations in the GALM gene which result in reduced activity of the enzyme galactose mutarotase. This enzyme catalyses the interconversion of the α- and ß-anomers of d-galactose and some other monosaccharides. Human galactose mutarotase is monomeric and its structure is largely composed of ß-sheets. The catalytic mechanism requires a histidine residue acting as an acid, and a glutamate acting as a base. Together, these residues open the pyranose ring of d-galactose enabling free rotation of the bond between the first two carbon atoms in the monosaccharide. This can cause reversal of the configuration of the hydroxyl group attached to carbon 1. Type IV galactosemia manifests with similar symptoms to type II galactosemia (galactokinase deficiency), i.e. early onset cataracts. However, as a recently discovered disease, the longer-term consequences are unknown. The physiological role, if any, of galactose mutarotase's reactions with other monosaccharides are not yet known. The possible associations with other proteins also require further investigation.

RNA interference dynamics in juvenile Fasciola hepatica are altered during in vitro growth and development.

For over a decade RNA interference (RNAi) has been an important molecular tool for functional genomics studies in parasitic flatworms. Despite this, our understanding of RNAi dynamics in many flatworm parasites, such as the temperate liver fluke (Fasciola hepatica), remains rudimentary. The ability to maintain developing juvenile fluke in vitro provides the opportunity to perform functional studies during development of the key pathogenic life stage. Here, we investigate the RNAi competence of developing juvenile liver fluke. Firstly, all life stages examined possess, and express, core candidate RNAi effectors encouraging the hypothesis that all life stages of F. hepatica are RNAi competent. RNAi effector analyses supported growing evidence that parasitic flatworms have evolved a separate clade of RNAi effectors with unknown function. Secondly, we assessed the impact of growth/development during in vitro culture on RNAi in F. hepatica juveniles and found that during the first week post-excystment liver fluke juveniles exhibit quantitatively lower RNAi mediated transcript knockdown when maintained in growth inducing media. This did not appear to occur in older in vitro juveniles, suggesting that rapidly shifting transcript dynamics over the first week following excystment alters RNAi efficacy after a single 24 h exposure to double stranded (ds)RNA. Finally, RNAi efficiency was found to be improved through use of a repeated dsRNA exposure methodology that has facilitated silencing of genes in a range of tissues, thereby increasing the utility of RNAi as a functional genomics tool in F. hepatica.

Destressing Yeast for Higher Biofuel Yields: Can Excess Chaotropicity Be Mitigated?

Biofuels have the capacity to contribute to carbon dioxide emission reduction and to energy security as oil reserves diminish and/or become concentrated in politically unstable regions. However, challenges exist in obtaining the maximum yield from industrial fermentations. One challenge arises from the nature of alcohols. These compounds are chaotropic (i.e. causes disorder in the system) which causes stress in the microbes producing the biofuel. Brewer's yeast (Saccharomyces cerevisiae) typically cannot grow at ethanol concentration much above 17% (v/v). Mitigation of these properties has the potential to increase yield. Previously, we have explored the effects of chaotropes on model enzyme systems and attempted (largely unsuccessfully) to offset these effects by kosmotropes (compounds which increase the order of the system, i.e. the "opposite" of chaotropes). Here we present some theoretical results which suggest that high molecular mass polyethylene glycols may be the most effective kosmotropic additives in terms of both efficacy and cost. The assumptions and limitations of these calculations are also presented. A deeper understanding of the effects of chaotropes on biofuel-producing microbes is likely to inform improvements in bioethanol yields and enable more rational approaches to the "neutralisation" of chaotropicity.

The Catalytic Cycle of the Antioxidant and Cancer-Associated Human NQO1 Enzyme: Hydride Transfer, Conformational Dynamics and Functional Cooperativity.

Human NQO1 [NAD(H):quinone oxidoreductase 1] is a multi-functional and stress-inducible dimeric protein involved in the antioxidant defense, the activation of cancer prodrugs and the stabilization of oncosuppressors. Despite its roles in human diseases, such as cancer and neurological disorders, a detailed characterization of its enzymatic cycle is still lacking. In this work, we provide a comprehensive analysis of the NQO1 catalytic cycle using rapid mixing techniques, including multiwavelength and spectral deconvolution studies, kinetic modeling and temperature-dependent kinetic isotope effects (KIEs). Our results systematically support the existence of two pathways for hydride transfer throughout the NQO1 catalytic cycle, likely reflecting that the two active sites in the dimer catalyze two-electron reduction with different rates, consistent with the cooperative binding of inhibitors such as dicoumarol. This negative cooperativity in NQO1 redox activity represents a sort of half-of-sites activity. Analysis of KIEs and their temperature dependence also show significantly different contributions from quantum tunneling, structural dynamics and reorganizations to catalysis at the two active sites. Our work will improve our understanding of the effects of cancer-associated single amino acid variants and post-translational modifications in this protein of high relevance in cancer progression and treatment.

In silico analysis of the effects of disease-associated mutations of ß-hexosaminidase A in Tay-Sachs disease.

Tay-Sachs disease (TSD), a deficiency of b-hexosaminidase A (Hex A), is a rare but debilitating hereditary metabolic disorder. Symptoms include extensive neurodegeneration and often result in death in infancy. We report an in silico study of 42 Hex A variants associated with the disease. Variants were separated into three groups according to the age of onset: infantile (n=28), juvenile (n=9) and adult (n=5). Protein stability, aggregation potential and the degree of conservation of residues were predicted using a range of in silico tools. We explored the relationship between these properties and the age of onset of TSD. There was no significant relationship between proteinstability and disease severity or between protein aggregation and disease severity. Infantile TSD had a significantly higher mean conservation score than nondisease associated variants. This was not seen in either juvenile or adult TSD. This study has established that the degree of residue conservation may be predictive of infantile TSD. It is possible that these more highly conserved residues are involved in trafficking of the protein to the lysosome. In addition, we developed and validated software tools to automate the process of in silico analysis of proteins involved in inherited metabolic diseases. Further work is required to identify the function of well-conserved residues to establish an in silico predictive model of TSD severity.

The roles and applications of chaotropes and kosmotropes in industrial fermentation processes.

Chaotropicity has long been recognised as a property of some compounds. Chaotropes tend to disrupt non-covalent interactions in biological macromolecules (e.g. proteins and nucleic acids) and supramolecular assemblies (e.g. phospholipid membranes). This results in the destabilisation and unfolding of these macromolecules and assemblies. Unsurprisingly, these compounds are typically harmful to living cells since they act against multiple targets, comprising cellular integrity and function. Kosmotropes are the opposite of chaotropes and these compounds promote the ordering and rigidification of biological macromolecules and assemblies. Since many biological macromolecules have optimum levels of flexibility, kosmotropes can also inhibit their activity and can be harmful to cells. Some products of industrial fermentations, most notably alcohols, are chaotropic. This property can be a limiting factor on rates of production and yields. It has been hypothesised that the addition of kosmotropes may mitigate the chaotropicity of some fermentation products. Some microbes naturally adapt to chaotropic environments by producing kosmotropic compatible solutes. Exploitation of this in industrial fermentations has been hampered by scientific and economic issues. The cost of the kosmotropes and their removal during purification needs to be considered. We lack a complete understanding of the chemistry of chaotropicity and a robust, quantitative framework for estimating overall chaotropicities of mixtures. This makes it difficult to predict the amount of kosmotrope required to neutralise the chaotropicity. This review considers examples of industrial fermentations where chaotropicity is an issue and suggests possible mitigations.

In Silico Analysis of the Effects of Point Mutations on α-Globin: Implications for α-Thalassemia.

Hemoglobinopathies are inherited diseases that impair the structure and function of the oxygen-carrying pigment hemoglobin (Hb). Adult Hb consists of two α and two ß subunits. α-Thalassemia (α-thal) affects the genes that code for the α-globin chains, HBA1 and HBA2. Mutations can result in asymptomatic, mild or severe outcomes depending on several factors, such as mutation type, number of mutations and the location at which they occur. PredictSNP was used to estimate whether every possible single nucleotide polymorphism (SNP) would have a neutral or deleterious effect on the protein. These results were then used to create a plot of predicted tolerance to change for each residue in the protein. Tolerance to change was negatively correlated with the residue's sequence conservation score. The PredictSNP data were compared to clinical reports of 110 selected variants in the literature. There were 29 disagreements between the two data types. Some of these could be resolved by considering the role of the affected residue in binding other molecules. The three-dimensional structures of some of these variant proteins were modeled. These models helped explain variants which affect heme binding. We predict that where a point mutation alters a residue that is intolerant to change, is well conserved and or involved in interactions, it is likely to be associated with disease. Overall, the data from this study could be used alongside biochemical and clinical data to assess novel α-globin variants.

Praziquantel: An Enigmatic, Yet Effective, Drug.

Praziquantel is a remarkably effective drug for the treatment of schistosomiasis. It has few side effects, some of which have been attributed to its inactive enantiomer. Few, if any, verified cases of drug resistance have been reported in a clinical setting. The preponderance of scientific evidence suggests that the drug works by dysregulating calcium homeostasis in the worm. Voltage-gated calcium channels have been proposed as the main pharmacological target of praziquantel, although no direct evidence of interaction with this protein is available. Here, the biochemical pharmacology of praziquantel is briefly reviewed and a hypothesis for its mechanism proposed. This hypothesis suggests that the drug works, in part, by disrupting an interaction between a voltage-gated calcium channel (SmCav1B) and an accessory protein, SmTAL1.

Therapies for galactosemia: a patent landscape.

Galactosemia is the inherited inability to metabolise galactose. The most common results from a lack of galactose 1-phosphate uridylyltransferase activity. The current treatment, removal of galactose from the diet, is inadequate and often fails to prevent long-term complications. Since 2015, three patents have been filed describing novel therapies. These are: the use of aldose reductase inhibitors to reduce cataracts and, possibly, other symptoms; salubrinal to stimulate cellular stress responses; mRNA therapy to increase cellular galactose 1-phosphate uridylyltransferase activity. The viability of all three is supported by academic studies. The potential and drawbacks of all three are discussed and evaluated.

Novel and selective inactivators of Triosephosphate isomerase with anti-trematode activity.

Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated 250 million people worldwide and the associated agricultural losses are estimated at more than US$ 6 billion per year. Current chemotherapy is limited. Triosephosphate isomerase (TIM), an enzyme of the glycolytic pathway, has emerged as a useful drug target in many parasites, including Fasciola hepatica TIM (FhTIM). We identified 21 novel compounds that selectively inhibit this enzyme. Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl-1,2,4-thiadiazole (compound 187) and FhTIM, which showed an IC50 of 5 µM and a Kd of 66 nM. In only 4 hours, this compound killed the juvenile form of F. hepatica with an IC50 of 3 µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC50 of 7 µM suggesting a previously uncharacterized role of FhTIM in the mechanism of action of this drug. Compound 187 was also active against various developmental stages of Schistosoma mansoni. The low toxicity in vitro in different cell types and lack of acute toxicity in mice was demonstrated for this compound, as was demonstrated the efficacy of 187 in vivo in F. hepatica infected mice. Finally, we obtained the first crystal structure of FhTIM at 1.9 Å resolution which allows us using docking to suggest a mechanism of interaction between compound 187 and TIM. In conclusion, we describe a promising drug candidate to control neglected trematode infections in human and animal health.

The Schistosoma mansoni tegumental allergen protein, SmTAL1: Binding to an IQ-motif from a voltage-gated ion channel and effects of praziquantel.

SmTAL1 is a calcium binding protein from the parasitic worm, Schistosoma mansoni. Structurally it is comprised of two domains - an N-terminal EF-hand domain and a C-terminal dynein light chain (DLC)-like domain. The protein has previously been shown to interact with the anti-schistosomal drug, praziquantel (PZQ). Here, we demonstrated that both EF-hands in the N-terminal domain are functional calcium ion binding sites. The second EF-hand appears to be more important in dictating affinity and mediating the conformational changes which occur on calcium ion binding. There is positive cooperativity between the four calcium ion binding sites in the dimeric form of SmTAL1. Both the EF-hand domain and the DLC-domain dimerise independently suggesting that both play a role in forming the SmTAL1 dimer. SmTAL1 binds non-cooperatively to PZQ and cooperatively to an IQ-motif from SmCav1B, a voltage-gated calcium channel. PZQ tends to strengthen this interaction, although the relationship is complex. These data suggest the hypothesis that SmTAL1 regulates at least one voltage-gated calcium channel and PZQ interferes with this process. This may be important in the molecular mechanism of this drug. It also suggests that compounds which bind SmTAL1, such as six from the Medicines for Malaria Box identified in this work, may represent possible leads for the discovery of novel antagonists.

The Mechanism of Action of Praziquantel: Can New Drugs Exploit Similar Mechanisms?

Praziquantel (PZQ) is the drug of choice for treating infection with worms from the genus Schistosoma. The drug is effective, cheap and has few side effects. However, despite its use in millions of patients for over 40 years its molecular mechanism of action remains elusive. Early studies demonstrated that PZQ disrupts calcium ion homeostasis in the worm and the current consensus is that it antagonises voltage-gated calcium channels. It is hypothesised that disruption of these channels results in uncontrolled calcium ion influx leading to uncontrolled muscle contraction and paralysis. However, other experimental studies have suggested a role for myosin regulatory light chains and adenosine uptake in the drug's mechanism of action. Assuming voltage-gated calcium channels do represent the main molecular target of PZQ, the precise binding site for the drug remains to be identified. Unlike other commonly used anti-parasitic drugs, there are few definitive reports of resistance to PZQ in the literature. The lack of knowledge about PZQ's molecular mechanism(s) undermines our ability to predict how resistance might arise and also hinder our attempts to develop alternative antischistosomal drugs which exploit the same target(s). Some PZQ derivatives have been identified which also kill or paralyse schistosomes in culture. However, none of these are in widespread clinical use. There is a pressing need for fundamental research into the molecular mechanism( s) of action of PZQ. Such research would enable new avenues for antischsistosomal drug discovery.

New model for the interaction of IQGAP1 with CDC42 and RAC1.

The specific and rapid formation of protein complexes, involving IQGAP family proteins, is essential for diverse cellular processes, such as adhesion, polarization, and directional migration. Although CDC42 and RAC1, prominent members of the RHO GTPase family, have been implicated in binding to and activating IQGAP1, the exact nature of this protein-protein recognition process has remained obscure. Here, we propose a mechanistic framework model that is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of cellular pathways.

A Dynamic Core in Human NQO1 Controls the Functional and Stability Effects of Ligand Binding and Their Communication across the Enzyme Dimer.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson's and Alzheimer´s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding of this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer, with FAD and dicoumarol binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, which is valuable for understanding at the molecular level the effects of disease-associated variants, post-translational modifications and ligand binding cooperativity in NQO1.