Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles.

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.

S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine ß-Synthase.

Cystathionine ß-synthase (CBS) is a key enzyme in human (patho)physiology with a central role in hydrogen sulfide metabolism. The enzyme is composed of a pyridoxal 5'-phosphate-binding catalytic domain, flanked by the following two domains: a heme-binding N-terminal domain and a regulatory C-terminal domain binding S-adenosyl-l-methionine (AdoMet). CO or NO(•) binding at the ferrous heme negatively modulates the enzyme activity. Conversely, AdoMet binding stimulates CBS activity. Here, we provide experimental evidence for a functional communication between the two domains. We report that AdoMet binding significantly enhances CBS inhibition by CO. Consistently, we observed increased affinity (∼5-fold) and faster association (∼10-fold) of CO to the ferrous heme at physiological AdoMet concentrations. NO(•) binding to reduced CBS was also enhanced by AdoMet, although to a lesser extent (∼2-fold higher affinity) as compared with CO. Importantly, CO and NO(•) binding was unchanged by AdoMet in a truncated form of CBS lacking the C-terminal regulatory domain. These unprecedented observations demonstrate that CBS activation by AdoMet puzzlingly sensitizes the enzyme toward inhibition by exogenous ligands, like CO and NO(•). This further supports the notion that CBS regulation is a complex process, involving the concerted action of multiple physiologically relevant effectors.

Small aminothiol compounds improve the function of Arg to Cys variant proteins: effect on the human cystathionine ß-synthase p.R336C.

The key regulatory point of L-methionine (Met) and L-homocysteine (Hcy) degradation is catalyzed by cystathionine beta-synthase (CBS). CBS deficiency is caused by mutations in CBS gene, often resulting in protein misfolding. The prevalence of CBS deficiency in Qatar is 1/1800, ∼200-fold higher than the worldwide prevalence of 1/344 000. Almost all patients bear the CBS p.R336C variant. More than 20 years ago, it was shown in vitro that two unrelated protein variants with a substitution of an arginine (Arg) residue by cysteine (Cys) could be rescued by cysteamine (mercaptoethylamine), likely via formation of a disulfide between Cys and cysteamine, functionally mimicking the wild-type (WT) Arg side-chain. Based on these findings, we aimed to study whether cysteamine was able to improve the function of p.R336C CBS variant. Additionally, we tested the effect of mercaptoethylguanidine (MEG), a compound with a guanidino and a thiol function that may resemble Arg structure better than cysteamine. Three purified recombinant CBS proteins (p.R336C, p.R336H and WT) were pre-incubated with cysteamine, MEG or Cys (as negative control), and CBS activity and stability were measured. Pre-incubation with cysteamine and MEG increased the enzymatic activity of the p.R336C protein, which was absent upon pre-incubation with Cys. The WT and the p.R336H variant enzyme activity presented no increase with any of the tested compounds. Our results show that cysteamine and MEG are able to specifically improve the function of the CBS p.R336C variant, suggesting that any Arg-to-Cys substitution accessible to these small molecules may be converted back to a moiety resembling Arg.

Unveiling the Pathogenic Molecular Mechanisms of the Most Common Variant (p.K329E) in Medium-Chain Acyl-CoA Dehydrogenase Deficiency by in Vitro and in Silico Approaches.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common genetic disorder affecting the mitochondrial fatty acid ß-oxidation pathway. The mature and functional form of human MCAD (hMCAD) is a homotetramer assembled as a dimer of dimers (monomers A/B and C/D). Each monomer binds a FAD cofactor, necessary for the enzyme's activity. The most frequent mutation in MCADD results from the substitution of a lysine with a glutamate in position 304 of mature hMCAD (p.K329E in the precursor protein). Here, we combined in vitro and in silico approaches to assess the impact of the p.K329E mutation on the protein's structure and function. Our in silico results demonstrated for the first time that the p.K329E mutation, despite lying at the dimer-dimer interface and being deeply buried inside the tetrameric core, seems to affect the tetramer surface, especially the ß-domain that forms part of the catalytic pocket wall. Additionally, the molecular dynamics data indicate a stronger impact of the mutation on the protein's motions in dimer A/B, while dimer C/D remains similar to the wild type. For dimer A/B, severe disruptions in the architecture of the pockets and in the FAD and octanoyl-CoA binding affinities were also observed. The presence of unaffected pockets (C/D) in the in silico studies may explain the decreased enzymatic activity determined for the variant protein (46% residual activity). Moreover, the in silico structural changes observed for the p.K329E variant protein provide an explanation for the structural instability observed experimentally, namely, the disturbed oligomeric profile, thermal stability, and conformational flexibility, with respect to the wild-type.

NO* binds human cystathionine ß-synthase quickly and tightly.

The hexa-coordinate heme in the H2S-generating human enzyme cystathionine ß-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO(•) or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO(•) was reported to bind at the CBS heme with very low affinity (Kd = 30-281 µm). This discrepancy was herein reconciled by investigating the NO(•) reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO(•) binds tightly to the ferrous CBS heme, with an apparent Kd ≤ 0.23 µm. In line with this result, at 25 °C, NO(•) binds quickly to CBS (k on ∼ 8 × 10(3) m(-1) s(-1)) and dissociates slowly from the enzyme (k off ∼ 0.003 s(-1)). The observed rate constants for NO(•) binding were found to be linearly dependent on [NO(•)] up to ∼ 800 µm NO(•), and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO(•) quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO(•). The novel findings reported here shed new light on CBS regulation by NO(•) and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO(•), CO, and H2S in cell signaling.

Insights into the regulatory domain of cystathionine Beta-synthase: characterization of six variant proteins.

Cystathionine beta-synthase (CBS) catalyzes the formation of cystathionine from homocysteine and serine. CBS is allosterically activated by S-adenosylmethionine (SAM), which binds to its C-terminal regulatory domain. Mutations in this domain lead to variants with high residual activity but lacking SAM activation. We characterized six C-terminal CBS variants (p.P427L, p.D444N, p.V449G, p.S500L, p.K523Sfs*18, and p.L540Q). To understand the effect of C-terminal mutations on the functional/structural properties of CBS, we performed dynamic light scattering, differential scanning fluorimetry, limited proteolysis, enzymatic characterization, and determination of SAM-binding affinity. Kinetic data confirm that the enzymatic function of these variants is not impaired. Although lacking SAM activation, the p.P427L and p.S500L were able to bind SAM at a lower extent than the wild type (WT), confirming that SAM binding and activation can be two independent events. At the structural level, the C-terminal variants presented various effects, either showing catalytic core instability and increased susceptibility toward aggregation or presenting with similar or higher stability than the WT. Our study highlights as the common feature to the C-terminal variants an impaired binding of SAM and no increase in enzymatic activity with physiological concentrations of the activator, suggesting the loss of regulation by SAM as a potential pathogenic mechanism.

Reduced response of Cystathionine Beta-Synthase (CBS) to S-Adenosylmethionine (SAM): Identification and functional analysis of CBS gene mutations in Homocystinuria patients.

A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs*2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients' fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria.

Real-time detection of enzymatically formed hydrogen sulfide by pathogenic variants of cystathionine beta-synthase using hemoglobin I of Lucina pectinata as a biosensor.

Classical homocystinuria is a rare disease caused by mutations in cystathionine ß-synthase (CBS) gene (OMIM 613381). CBS catalyzes the first step of the transsulfuration pathway that converts homocysteine (Hcy) into cystathionine (Cysta) via a number of co-substrates and mechanisms. Formation of Cysta by condensation of Hcy and cysteine (Cys) produces a molar equivalent of hydrogen sulfide (H2S). H2S plays important roles in cognitive and vascular functions. Clinically, patients with CBS deficiency present with vascular, ocular, neurological and skeletal impairments. Biochemically, CBS deficiency manifests with elevated Hcy and reduced concentration of Cysta in plasma and urine. A number of pathogenic variants of human CBS have been characterized by their residual enzymatic activity, but very few studies have examined H2S production by pathogenic CBS variants, possibly due to technical hurdles in H2S detection and quantification. We describe a method for the real-time, continuous quantification of H2S formed by wild-type and pathogenic variants of human recombinant CBS, as well as by fibroblast extracts from healthy controls and patients diagnosed with CBS deficiency. The method takes advantage of the specificity and high affinity of hemoglobin I of the clam Lucina pectinata toward H2S and is based on UV-visible spectrophotometry. Comparison with the gold-standard, end-point H2S quantification method employing monobromobimane, as well as correlations with CBS enzymatic activity determined by LC-MS/MS showed agreement and correlation, and permitted the direct, time-resolved determination of H2S production rates by purified human recombinant CBS and by CBS present in fibroblast extracts. Rates of H2S production were highest for wild-type CBS, and lower for pathogenic variants. This method enables the examination of structural determinants of CBS that are important for H2S production and its possible relevance to the clinical outcome of patients.

Isothermal denaturation fluorimetry vs differential scanning fluorimetry as tools for screening of stabilizers for protein freeze-drying: Human phenylalanine hydroxylase as the case study.

The structural maintenance of therapeutic proteins during formulation and/or storage is a critical aspect, particularly for multi-domain and/or multimeric proteins which usually exhibit intrinsic structural dynamics leading to aggregation with concomitant loss-of-function. Protein freeze-drying is a widely used technique to preserve protein structure and function during storage. To minimize chemical/physical stresses occurring during this process, protein stabilizers are usually included, their effect being strongly dependent on the target protein. Therefore, they should be screened for on a time-consuming case-by-case basis. Herein, differential scanning fluorimetry (DSF) and isothermal denaturation fluorimetry (ITDF) were employed to screen, among different classes of freeze-drying additives, for the most effective stabilizer of the model protein human phenylalanine hydroxylase (hPAH). Correlation studies among retrieved DSF and ITDF parameters with recovered enzyme amount and activity indicated ITDF as the most appropriate screening method. Biochemical and biophysical characterization of hPAH freeze-dried with ITDF-selected stabilizers and a long-term storage study (12 months, 5 ± 3 °C) showed that the selected compounds prevented protein aggregation and preserved hPAH structural and functional properties throughout time storage. Our results provide a solid basis towards the choice of ITDF as a high-throughput screening step for the identification of protein freeze-drying protectors.

Metabolism-Guided Optimization of Tryptophanol-Derived Isoindolinone p53 Activators.

For the first time, the pharmacokinetic (PK) profile of tryptophanol-derived isoindolinones, previously reported as p53 activators, was investigated. From the metabolites' identification, performed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS), followed by their preparation and structural elucidation, it was possible to identify that the indole C2 and C3 are the main target of the cytochrome P450 (CYP)-promoted oxidative metabolism in the tryptophanol-derived isoindolinone scaffold. Based on these findings, to search for novel p53 activators a series of 16 enantiopure tryptophanol-derived isoindolinones substituted with a bromine in indole C2 was prepared, in yields of 62-89%, and their antiproliferative activity evaluated in human colon adenocarcinoma HCT116 cell lines with and without p53. Structural optimization led to the identification of two (S)-tryptophanol-derived isoindolinones 3.9-fold and 1.9-fold more active than hit SLMP53-1, respectively. Compounds' metabolic stability evaluation revealed that this substitution led to a metabolic switch, with the impact of Phase I oxidative metabolism being minimized. Through differential scanning fluorimetry (DSF) experiments, the most active compound of the series in cell assays led to an increase in the protein melting temperature (Tm) of 10.39 °C, suggesting an effective binding to wild-type p53 core domain.

Functional and structural impact of 10 ACADM missense mutations on human medium chain acyl-Coa dehydrogenase.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is associated with ACADM gene mutations, leading to an impaired function and/or structure of MCAD. Importantly, after import into the mitochondria, MCAD must incorporate a molecule of flavin adenine dinucleotide (FAD) per subunit and assemble into tetramers. However, the effect of MCAD amino acid substitutions on FAD incorporation has not been investigated. Herein, the commonest MCAD variant (p.K304E) and 11 additional rare variants (p.Y48C, p.R55G, p.A88P, p.Y133C, p.A140T, p.D143V, p.G224R, p.L238F, p.V264I, p.Y372N, and p.G377V) were functionally and structurally characterized. Half of the studied variants presented a FAD content <65 % compared to the wild-type. Most of them were recovered as tetramers, except the p.Y372N (mainly as dimers). No correlation was found between the levels of tetramers and FAD content. However, a correlation between FAD content and the cofactor's affinity, proteolytic stability, thermostability, and thermal inactivation was established. We showed that the studied amino acid changes in MCAD may alter the substrate chain-length dependence and the interaction with electron-transferring-flavoprotein (ETF) necessary for a proper functioning electron transfer thus adding additional layers of complexity to the pathological effect of ACADM missense mutations. Although the majority of the variant MCADs presented an impaired capacity to retain FAD during their synthesis, some of them were structurally rescued by cofactor supplementation, suggesting that in the mitochondrial environment the levels and activity of those variants may be dependent of FAD's availability thus contributing for the heterogeneity of the MCADD phenotype found in patients presenting the same genotype.

Heterotetrameric forms of human phenylalanine hydroxylase: co-expression of wild-type and mutant forms in a bicistronic system.

Hybrid forms of human phenylalanine hydroxylase (hPAH) mutants have been found to present catalytic activities lower than predicted from the individual recombinant forms, indicating that interallelic complementation could be a major determinant of the metabolic phenotype of compound heterozygous phenylketonuric (PKU) patients. To provide a molecular explanation for interallelic complementation we have here developed a bicistronic expression system and a purification strategy to obtain isolated hPAH heteromeric forms. On co-expression of WT-hPAH (~50% tetramer; ~10% dimer) and the N- and C-terminally truncated form ΔN102/ΔC24-hPAH (~80% dimer) no heterodimers were recovered. Moreover, by co-expression of WT-hPAH and the N-terminally truncated form ΔN102-hPAH (~95% tetramer), heterotetramers, as a result of an assembly of two different homodimers, were isolated. The recovered (WT)/(ΔN102)-hPAH heterotetramers revealed a catalytic activity deviating significantly from that calculated by averaging the respective recombinant homotetrameric forms. The heterotetramer assembly also results in conformational changes in the WT-hPAH protomer, as detected by trypsin limited proteolysis. The finding that the presence of two homodimers with different kinetic parameters influences the properties of the resulting heterotetrameric protein indicates that the dimers exhibit interactions which are transmitted across the assembled tetramer. The bicistronic expression system developed here allowed the isolation of hybrid forms that exhibit negative interallelic complementation, and may represent a model system for studying the molecular pathogenic mechanisms of PAH gene mutations in compound heterozygous PKU patients, providing the rationale to understand the observed inconsistencies both in genotype/phenotype correlations and in the response to BH(4) supplementation.

Phenylketonuria as a protein misfolding disease: The mutation pG46S in phenylalanine hydroxylase promotes self-association and fibril formation.

The missense mutation pG46S in the regulatory (R) domain of human phenylalanine hydroxylase (hPAH), associated with a severe form of phenylketonuria, generates a misfolded protein which is rapidly degraded on expression in HEK293 cells. When overexpressed as a MBP-G46S fusion protein, soluble and fully active tetrameric/dimeric forms are assembled and recovered in a metastable conformational state. When MBP is cleaved off, G46S undergoes a conformational change and self-associates with a lag phase and an autocatalytic growth phase (tetramers≫dimers), as determined by light scattering. The self-association is controlled by pH, ionic strength, temperature, protein concentration and the phosphorylation state of Ser16; the net charge of the protein being a main modulator of the process. A superstoichiometric amount of WT dimers revealed a 2-fold enhancement of the rate of G46S dimer self-association. Electron microscopy demonstrates the formation of higher-order oligomers and linear polymers of variable length, partly as a branching network, and partly as individual long and twisted fibrils (diameter ~145-300Å). The heat-shock proteins Hsp70/Hsp40, Hsp90 and a proposed pharmacological PAH chaperone (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) partly inhibit the self-association process. Our data indicate that the G46S mutation results in a N-terminal extension of α-helix 1 which perturbs the wild-type α-ß sandwich motif in the R-domain and promotes new intermolecular contacts, self-association and non-amyloid fibril formation. The metastable conformational state of G46S as a MBP fusion protein, and its self-association propensity when released from MBP, may represent a model system for the study of other hPAH missense mutations characterized by misfolded proteins.

Formulation of spray dried enzymes for dry powder inhalers: An integrated methodology.

One of the main challenges when developing a spray dried formulation of an inhalable enzyme is the generation of a safe and effective aerosol, able to reach the lungs, while preserving protein function and structural levels of the biologic. Hence, an appropriate excipient selection based on enzyme stabilization, inhalation precedence and spray drying (SD) process development is required to meet this balance. Herein, an integrated methodology is presented to expedite the selection of the best dry powder inhaler excipient system to formulate three model enzymes of increasing molecular mass and structural complexity belonging to the oxidoreductase class and often implicated in oxidative stress: superoxide dismutase, glucose oxidase and catalase. Three non-reducing sugars and four amino acids were screened using High Throughput Isothermal Denaturation Fluorimetry (HT-ITDF) for a stabilizing effect on the enzymes quaternary structure. For each tested enzyme, the sugar and amino acid showcasing a stabilizing effect, were spray dried together at fixed process conditions for three different ratios, to assess which formulation would then display the best aerodynamic performance. After SD, using the selected conditions, all powders displayed 65-85% of fine particle fraction (FPF) whilst each enzyme kept the oligomeric state. The present integrated methodology proved to be successful, allowing to narrow down 36 potential formulations (three sugars × four amino acids × three ratios) to only one for each enzyme, within few hours, while requiring a µg range of sample amount.

Impact of Fluorinated Ionic Liquids on Human Phenylalanine Hydroxylase-A Potential Drug Delivery System.

Phenylketonuria (PKU) is an autosomal recessive disease caused by deficient activity of human phenylalanine hydroxylase (hPAH), which can lead to neurologic impairments in untreated patients. Although some therapies are already available for PKU, these are not without drawbacks. Enzyme-replacement therapy through the delivery of functional hPAH could be a promising strategy. In this work, biophysical methods were used to evaluate the potential of [N1112(OH)][C4F9SO3], a biocompatible fluorinated ionic liquid (FIL), as a delivery system of hPAH. The results herein presented show that [N1112(OH)][C4F9SO3] spontaneously forms micelles in a solution that can encapsulate hPAH. This FIL has no significant effect on the secondary structure of hPAH and is able to increase its enzymatic activity, despite the negative impact on protein thermostability. The influence of [N1112(OH)][C4F9SO3] on the complex oligomerization equilibrium of hPAH was also assessed.

Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeation.

In general, erythrocytes are highly permeable to water, urea and glycerol. However, expression of aquaporin isoforms in erythrocytes appears to be species characteristic. In the present study, human (hRBC) and bovine (bRBC) erythrocytes were chosen for comparative studies due to their significant difference in membrane glycerol permeability. Osmotic water permeability (P(f)) at 23°C was (2.89 ± 0.37) × 10(-2) and (5.12 ± 0.61) × 10(-2)cms(-1) for human and bovine cells, respectively, with similar activation energies for water transport. Glycerol permeability (P(gly)) for human ((1.37 ± 0.26) × 10(-5)cms(-1)) differed in three orders of magnitude from bovine erythrocytes ((5.82 ± 0.37) × 10(-8)cms(-1)) that also showed higher activation energy for glycerol transport. When compared to human, bovine erythrocytes showed a similar expression pattern of AQP1 glycosylated forms on immunoblot analysis, though in slight higher levels, which could be correlated with the 1.5-fold larger P(f) found. However, AQP3 expression was not detectable. Immunofluorescence analysis confirmed the absence of AQP3 expression in bovine erythrocyte membranes. In conclusion, lack of AQP3 in bovine erythrocytes points to the lipid pathway as responsible for glycerol permeation and explains the low glycerol permeability and high E(a) for transport observed in ruminants.

The G46S-hPAH mutant protein: a model to study the rescue of aggregation-prone PKU mutations by chaperones.

Phenylketonuria (PKU), the most common inborn error of metabolism, is caused by dysfunction of the liver enzyme phenylalanine hydroxylase (PAH), with more than 550 PAH gene mutations identified to date. A large number of these mutations result in mutant forms of the enzyme displaying reduced stability, increased propensity to aggregate, and accelerated in cellulo degradation. Loss or reduction of human PAH activity results in hyperphenylalaninemia (HPA) which, if untreated, results in severe mental retardation and impaired cognitive development. Until now, strict low phenylalanine diet has been the most effective therapy, but as a protein misfolding disease PKU is a good candidate for treatment by natural/chemical/pharmacological chaperones. The natural cofactor of human PAH, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), has already been approved for oral treatment of HPA, giving a positive response in mild forms of the disease showing considerable residual enzymatic activity. In the case of the most severe forms of PKU, ongoing studies with chemical and pharmacological chaperones to rescue misfolded mutant proteins from aggregation and degradation are providing promising results. The PKU mutation G46S is associated with a severe form of the disease, resulting in an aggregation-prone protein. The human PAH mutant G46S is rapidly degraded in the cellular environment and, in vitro (upon removal of its stabilizing fusion partner maltose binding protein (MBP)) self-associates to form higher-order oligomers/fibrils. Here, we present an in vitro experimental model system to study the modulation of G46S aggregation by chemical/pharmacological chaperones, which may represent a useful approach to study the rescue of other severe PKU mutations by chemical/pharmacological chaperones.

Engineering Organoids for in vitro Modeling of Phenylketonuria.

Phenylketonuria is a recessive genetic disorder of amino-acid metabolism, where impaired phenylalanine hydroxylase function leads to the accumulation of neurotoxic phenylalanine levels in the brain. Severe cognitive and neuronal impairment are observed in untreated/late-diagnosed patients, and even early treated ones are not safe from life-long sequelae. Despite the wealth of knowledge acquired from available disease models, the chronic effect of Phenylketonuria in the brain is still poorly understood and the consequences to the aging brain remain an open question. Thus, there is the need for better predictive models, able to recapitulate specific mechanisms of this disease. Human induced pluripotent stem cells (hiPSCs), with their ability to differentiate and self-organize in multiple tissues, might provide a new exciting in vitro platform to model specific PKU-derived neuronal impairment. In this review, we gather what is known about the impact of phenylalanine in the brain of patients and highlight where hiPSC-derived organoids could contribute to the understanding of this disease.

The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity.

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

In Silico and In Vitro Tailoring of a Chitosan Nanoformulation of a Human Metabolic Enzyme.

Enzyme nanoencapsulation holds an enormous potential to develop new therapeutic approaches to a large set of human pathologies including cancer, infectious diseases and inherited metabolic disorders. However, enzyme formulation has been limited by the need to maintain the catalytic function, which is governed by protein conformation. Herein we report the rational design of a delivery system based on chitosan for effective encapsulation of a functionally and structurally complex human metabolic enzyme through ionic gelation with tripolyphosphate. The rationale was to use a mild methodology to entrap the multimeric multidomain 200 kDa human phenylalanine hydroxylase (hPAH) in a polyol-like matrix that would allow an efficient maintenance of protein structure and function, avoiding formulation stress conditions. Through an in silico and in vitro based development, the particulate system was optimized with modulation of nanomaterials protonation status, polymer, counterion and protein ratios, taking into account particle size, polydispersity index, surface charge, particle yield production, protein free energy of folding, electrostatic surface potential, charge, encapsulation efficiency, loading capacity and transmission electron microscopy morphology. Evaluation of the thermal stability, substrate binding profile, relative enzymatic activity, and substrate activation ratio of the encapsulated hPAH suggests that the formulation procedure does not affect protein stability, allowing an effective maintenance of hPAH biological function. Hence, this study provides an important framework for an enzyme formulation process.