Cellular prion protein neither binds to alpha-synuclein oligomers nor mediates their detrimental effects.

α-Synuclein oligomers are crucial players in the pathogenesis of Parkinson's disease. Some mechanisms involved in α-synuclein oligomer detrimental effects include membrane damage, neuroinflammation and protein-protein interactions. Recently, the cellular prion protein (PrPC) emerged as an interactor of α-synuclein oligomers, apparently mediating their detrimental activities. Through direct in vivo and in vitro approaches we herein investigated the existence of a direct cross-talk between α-synuclein oligomers and PrPC. In vitro, we assessed α-synuclein oligomer toxicity by comparing the effect in Prnp+/+ versus PrPC knockout (Prnp0/0) hippocampal neurons. Through an in vivo acute mouse model, where α-synuclein oligomers injected intracerebroventricularly induce memory impairment and neuroinflammation, we verified whether these detrimental effects were preserved in Prnp0/0 mice. In addition, PrPC-α-synuclein oligomer direct binding was investigated through surface plasmon resonance. We found that PrPC was not mandatory to mediate α-synuclein oligomer detrimental effects in vitro or in vivo. Indeed, α-synuclein oligomer toxicity was comparable in Prnp+/+ and Prnp0/0 neurons and both Prnp+/+ and Prnp0/0 mice injected with α-synuclein oligomers displayed memory deficit and hippocampal gliosis. Moreover, surface plasmon resonance analyses ruled out PrPC-α-synuclein oligomer binding. Our findings indicate that PrPC neither binds α-synuclein oligomers nor mediates their detrimental actions. Therefore, it is likely that PrPC-dependent and PrPC-independent pathways co-exist in Parkinson's disease.

Human d-amino acid oxidase: The inactive G183R variant.

In the brain, the enzyme d-amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-serine, a main positive modulator of the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR). Dysregulation in d-serine signaling is implicated in the NMDAR dysfunctions observed in various brain diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, schizophrenia. A strain of ddY mice lacking DAAO activity due to the G181R substitution (DAAOG181R mice) and exhibiting increased d-serine concentration as compared to wild-type mice shows altered pain response, improved adaptative learning and cognitive functions, and larger hippocampal long-term potentiation. In past years, this mice line has been used to shed light on physiological and pathological brain functions related to NMDAR. Here, we decided to introduce the corresponding substitution in human DAAO (hDAAO). The recombinant G183R hDAAO is produced as an inactive apoprotein: the substitution alters the protein conformation that negatively affects the ability to bind the flavin cofactor in the orientation required for hydride-transfer during catalysis. At the cellular level, the overexpressed G183R hDAAO is not fully targeted to peroxisomes, forms protein aggregates showing a strong colocalization with ubiquitin, and significantly (7-fold) increases both the d-serine cellular concentration and the D/(D+L)-serine ratio. Taken together, our investigation warrants caution in using DAAOG181R mice: the abolition of enzymatic activity is coupled to DAAO aggregation, a central process in different pathological conditions. The effect due to G181R substitution in DAAO could be misleading: the effects due to impairment of d-serine degradation overlap with those related to aggregates accumulation.

Alpha-synuclein oligomers impair memory through glial cell activation and via Toll-like receptor 2.

Alpha-synuclein oligomers (α-synOs) are emerging as crucial factors in the pathogenesis of synucleinopathies. Although the connection between neuroinflammation and α-syn still remains elusive, increasing evidence suggests that extracellular moieties activate glial cells leading to neuronal damage. Using an acute mouse model, we explored whether α-synOs induce memory impairment in association to neuroinflammation, addressing Toll-like receptors 2 and 4 (TLR2 and TLR4) involvement. We found that α-synOs abolished mouse memory establishment in association to hippocampal glial activation. On brain slices α-synOs inhibited long-term potentiation. Indomethacin and Ibuprofen prevented the α-synOs-mediated detrimental actions. Furthermore, while the TLR2 functional inhibitor antibody prevented the memory deficit, oligomers induced memory deficits in the TLR4 knockout mice. In conclusion, solely α-synOs induce memory impairment likely inhibiting synaptic plasticity. α-synOs lead to hippocampal gliosis that is involved in memory impairment. Moreover, while the oligomer-mediated detrimental actions are TLR2 dependent, the involvement of TLR4 was ruled out.

Structure-function relationships in human d-amino acid oxidase variants corresponding to known SNPs.

In the brain, d-amino acid oxidase plays a key role in modulating the N-methyl-d-aspartate receptor (NMDAR) activation state, catalyzing the stereospecific degradation of the coagonist d-serine. A relationship between d-serine signaling deregulation, NMDAR dysfunction, and CNS diseases is presumed. Notably, the R199W substitution in human DAAO (hDAAO) was associated with familial amyotrophic lateral sclerosis (ALS), and further coding substitutions, i.e., R199Q and W209R, were also deposited in the single nucleotide polymorphism database. Here, we investigated the biochemical properties of these different hDAAO variants. The W209R hDAAO variant shows an improved d-serine degradation ability (higher activity and affinity for the cofactor FAD) and produces a greater decrease in cellular d/(d+l) serine ratio than the wild-type counterpart when expressed in U87 cells. The production of H2O2 as result of excessive d-serine degradation by this hDAAO variant may represent the factor affecting cell viability after stable transfection. The R199W/Q substitution in hDAAO altered the protein conformation and enzymatic activity was lost under conditions resembling the cellular ones: this resulted in an abnormal increase in cellular d-serine levels. Altogether, these results indicate that substitutions that affect hDAAO functionality directly impact on d-serine cellular levels (at least in the model cell system used). The pathological effect of the expression of the R199W hDAAO, as observed in familial ALS, originates from both protein instability and a decrease in kinetic efficiency: the increase in synaptic d-serine may be mainly responsible for the neurotoxic effect. This information is expected to drive future targeted treatments.

Characterization of human DAAO variants potentially related to an increased risk of schizophrenia.

Considering the key role of d-serine in N-methyl-d-aspartate receptor-mediated neurotransmission, it is highly relevant to define the role that enzymes play in d-serine synthesis and degradation. In particular, the details of regulation of the d-serine catabolic human enzyme d-amino acid oxidase (hDAAO) are unknown although different lines of evidence have shown it to be involved in schizophrenia susceptibility. Here we investigated the effect of three single nucleotide polymorphisms and known mutations in hDAAO, i.e., D31H, R279A, and G331V. A very low amount of soluble G331V hDAAO is produced in E. coli cells: the recombinant variant enzyme is fully active. Human U87 glioblastoma cells transiently transfected for G331V hDAAO show a low viability, a significant amount of protein aggregates, and augmented apoptosis. The recombinant D31H and R279A hDAAO variants do not show alterations in tertiary and quaternary structures, thermal stability, binding affinity for inhibitors, and the modulator pLG72, whereas the kinetic efficiency and the affinity for d-serine and for FAD were higher than for the wild-type enzyme. While these effects for the substitution at position 31 cannot be structurally explained, the R279A mutation might affect the hDAAO FAD-binding affinity by altering the "structurally ambivalent" peptide V47-L51. In agreement with the observed increased activity, expression of D31H and R279A hDAAO variants in U87 cells produces a higher decrease in cellular d/(d+l) serine ratio than the wild-type counterpart. In vivo, these substitutions could affect cellular d-serine concentration and its release at synapsis and thus might be relevant for schizophrenia susceptibility.

Biochemical and cellular studies of three human 3-phosphoglycerate dehydrogenase variants responsible for pathological reduced L-serine levels.

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches.

Effect of l-serine and magnesium ions on the functional properties of human phosphoserine phosphatase and its pathogenetic variants.

L-Ser supply in the central nervous system of mammals mostly relies on its endogenous biosynthesis by the phosphorylated pathway (PP). Defects in any of the three enzymes operating in the pathway result in a group of neurometabolic diseases collectively known as serine deficiency disorders (SDDs). Phosphoserine phosphatase (PSP) catalyzes the last, irreversible step of the PP. Here we investigated in detail the role of physiological modulators of human PSP activity and the properties of three natural PSP variants (A35T, D32N and M52T) associated with SDDs. Our results, partially contradicting previous reports, indicate that: i. PSP is almost fully saturated with Mg2+ under physiological conditions and fluctuations in Mg2+ and Ca2+ concentrations are unlikely to play a modulatory role on PSP activity; ii. Inhibition by L-Ser, albeit at play on the isolated PSP, does not exert any effect on the flux through the PP unless the enzyme activity is severely impaired by inactivating substitutions; iii. The so-far poorly investigated A35T substitution was the most detrimental, with a 50-fold reduction in catalytic efficiency, and a reduction in thermal stability (as well as an increase in the IC50 for L-Ser). The M52T substitution had similar, but milder effects, while the D32N variant behaved like the wild-type enzyme. iv. Predictions of the structural effects of the A35T and M52T substitutions with ColabFold suggest that they might affect the structure of the flexible helix-loop region.

Structural characterization of PHOX2B and its DNA interaction shed light on the molecular basis of the +7Ala variant pathogenicity in CCHS.

An expansion of poly-alanine up to +13 residues in the C-terminus of the transcription factor PHOX2B underlies the onset of congenital central hypoventilation syndrome (CCHS). Recent studies demonstrated that the alanine tract expansion influences PHOX2B folding and activity. Therefore, structural information on PHOX2B is an important target for obtaining clues to elucidate the insurgence of the alanine expansion-related syndrome and also for defining a viable therapy. Here we report by NMR spectroscopy the structural characterization of the homeodomain (HD) of PHOX2B and HD + C-terminus PHOX2B protein, free and in the presence of the target DNA. The obtained structural data are then exploited to obtain a structural model of the PHOX2B-DNA interaction. In addition, the variant +7Ala, responsible for one of the most frequent forms of the syndrome, was analysed, showing different conformational proprieties in solution and a strong propensity to aggregation. Our data suggest that the elongated poly-alanine tract would be related to disease onset through a loss-of-function mechanism. Overall, this study paves the way for the future rational design of therapeutic drugs, suggesting as a possible therapeutic route the use of specific anti-aggregating molecules capable of preventing variant aggregation and possibly restoring the DNA-binding activity of PHOX2B.

One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli.

BACKGROUND: Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson's disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. RESULTS: A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. CONCLUSIONS: To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson's disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures.

The D-amino acid oxidase-carbon nanotubes: evaluation of cytotoxicity and biocompatibility of a potential anticancer nanosystem.

The 'enzyme prodrug therapy' represents a promising strategy to overcome limitations of current cancer treatments by the systemic administration of prodrugs, converted by a foreign enzyme into an active anticancer compound directly in tumor sites. One example is D-amino acid oxidase (DAAO), a dimeric flavoenzyme able to catalyze the oxidative deamination of D-amino acids with production of hydrogen peroxide, a reactive oxygen species (ROS), able to favor cancer cells death. A DAAO variant containing five aminoacidic substitutions (mDAAO) was demonstrated to possess a better therapeutic efficacy under low O2 concentration than wild-type DAAO (wtDAAO). Recently, aiming to design promising nanocarriers for DAAO, multi-walled carbon nanotubes (MWCNTs) were functionalized with polyethylene glycol (PEG) to reduce their tendency to aggregation and to improve their biocompatibility. Here, wtDAAO and mDAAO were adsorbed on PEGylated MWCNTs and their activity and cytotoxicity were tested. While PEG-MWCNTs-DAAOs have shown a higher activity than pristine MWCNTs-DAAO (independently on the DAAO variant used), PEG-MWCNTs-mDAAO showed a higher cytotoxicity than PEG-MWCNTs-wtDAAO at low O2 concentration. In order to evaluate the nanocarriers' biocompatibility, PEG-MWCNTs-DAAOs were incubated in human serum and the composition of protein corona was investigated via nLC-MS/MS, aiming to characterize both soft and hard coronas. The mDAAO variant has influenced the bio-corona composition in both number of proteins and presence of opsonins and dysopsonins: notably, the soft corona of PEG-MWCNTs-mDAAO contained less proteins and was more enriched in proteins able to inhibit the immune response than PEG-MWCNTs-wtDAAO. Considering the obtained results, the PEGylated MWCNTs conjugated with the mDAAO variant seems a promising candidate for a selective antitumor oxidative therapy: under anoxic-like conditions, this novel drug delivery system showed a remarkable cytotoxic effect controlled by the substrate addition, against different tumor cell lines, and a bio-corona composition devoted to prolong its blood circulation time, thus improving the drug's biodistribution. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03568-1.

Structure-function relationships in human D-amino acid oxidase.

Since D-amino acids were identified in mammals, D-serine has been one of the most extensively studied "unnatural amino acids". This brain-enriched transmitter-like molecule plays a pivotal role in the human central nervous system by modulating the activity of NMDA receptors. Physiological levels of D-serine are required for normal brain development and function; thus, any alterations in neuromodulator concentrations might result in NMDA receptor dysfunction, which is known to be involved in several pathological conditions, including neurodegeneration(s), epilepsy, schizophrenia, and bipolar disorder. In the brain, the concentration of D-serine stored in cells is defined by the activity of two enzymes: serine racemase (responsible for both the synthesis and degradation) and D-amino acid oxidase (which catalyzes D-serine degradation). Both enzymes emerged recently as new potential therapeutic targets for NMDA receptor-related diseases. In this review we have focused on human D-amino acid oxidase and provide an extensive overview of the biochemical and structural properties of this flavoprotein and their functional significance. Furthermore, we discuss the mechanisms involved in modulating enzyme activity and stability with the aim to substantiate the pivotal role of D-amino acid oxidase in brain D-serine metabolism in physiological and pathological conditions and to highlight its great significance for novel drug design/development.

Recombinant production of eight human cytosolic aminotransferases and assessment of their potential involvement in glyoxylate metabolism.

PH1 (primary hyperoxaluria type 1) is a severe inborn disorder of glyoxylate metabolism caused by a functional deficiency of the peroxisomal enzyme AGXT (alanine-glyoxylate aminotransferase), which converts glyoxylate into glycine using L-alanine as the amino-group donor. Even though pre-genomic studies indicate that other human transaminases can convert glyoxylate into glycine, in PH1 patients these enzymes are apparently unable to compensate for the lack of AGXT, perhaps due to their limited levels of expression, their localization in an inappropriate cell compartment or the scarcity of the required amino-group donor. In the present paper, we describe the cloning of eight human cytosolic aminotransferases, their recombinant expression as His6-tagged proteins and a comparative study on their ability to transaminate glyoxylate, using any standard amino acid as an amino-group donor. To selectively quantify the glycine formed, we have developed and validated an assay based on bacterial GO (glycine oxidase); this assay allows the detection of enzymes that produce glycine by transamination in the presence of mixtures of potential amino-group donors and without separation of the product from the substrates. We show that among the eight enzymes tested, only GPT (alanine transaminase) and PSAT1 (phosphoserine aminotransferase 1) can transaminate glyoxylate with good efficiency, using L-glutamate (and, for GPT, also L-alanine) as the best amino-group donor. These findings confirm that glyoxylate transamination can occur in the cytosol, in direct competition with the conversion of glyoxylate into oxalate. The potential implications for the treatment of primary hyperoxaluria are discussed.

Molecular insights into the role of the polyalanine region in mediating PHOX2B aggregation.

About 90% of congenital central hypoventilation syndrome (CCHS) patients show polyalanine triplet expansions in the coding region of transcription factor PHOX2B, which renders this protein an intriguing target to understand the insurgence of this syndrome and for the design of a novel therapeutical approach. Consistently with the role of PHOX2B as a transcriptional regulator, it is reasonable that a general transcriptional dysregulation caused by the polyalanine expansion might represent an important mechanism underlying CCHS pathogenesis. Therefore, this study focused on the biochemical characterization of different PHOX2B variants, such as a variant containing the correct C-terminal (20 alanines) stretch, one of the most frequent polyalanine expansions (+7 alanines), and a variant lacking the complete alanine stretch (0 alanines). Comparison of the different variants by a multidisciplinary approach based on different methodologies (including circular dichroism, spectrofluorimetry, light scattering, and Atomic Force Microscopy studies) highlighted the propensity to aggregate for the PHOX2B variant containing the polyalanine expansion (+7-alanines), especially in the presence of DNA, while the 0-alanines variant resembled the protein with the correct polyalanine length. Moreover, and unexpectedly, the formation of fibrils was revealed only for the pathological variant, suggesting a plausible role of such fibrils in the insurgence of CCHS.

Relevance of the flavin binding to the stability and folding of engineered cholesterol oxidase containing noncovalently bound FAD.

The flavoprotein cholesterol oxidase (CO) from Brevibacterium sterolicum is a monomeric flavoenzyme containing one molecule of FAD cofactor covalently linked to His69. The elimination of the covalent link following the His69Ala substitution was demonstrated to result in a significant decrease in activity, in the midpoint redox potential of the flavin, and in stability with respect to the wild-type enzyme, but does not modify the overall structure of the enzyme. We used CO as a model system to dissect the changes due to the elimination of the covalent link between the flavin and the protein (by comparing the wild-type and H69A CO holoproteins) with those due to the elimination of the cofactor (by comparing the holo- and apoprotein forms of H69A CO). The apoprotein of H69A CO lacks the characteristic tertiary structure of the holoprotein and displays larger hydrophobic surfaces; its urea-induced unfolding does not occur by a simple two-state mechanism and is largely nonreversible. Minor alterations in the flavin binding region are evident between the native and the refolded proteins, and are likely responsible for the low refolding yield observed. A model for the equilibrium unfolding of H69A CO that also takes into consideration the effects of cofactor binding and dissociation, and thus may be of general significance in terms of the relationships between cofactor uptake and folding in flavoproteins, is presented.

Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach.

D-amino acid oxidase (DAAO) is a FAD-containing flavoprotein that dehydrogenates the D-isomer of amino acids to the corresponding imino acids, coupled with the reduction of FAD. The cofactor then reoxidizes on molecular oxygen and the imino acid hydrolyzes spontaneously to the alpha-keto acid and ammonia. In vitro DAAO displays broad substrate specificity, acting on several neutral and basic D-amino acids: the most efficient substrates are amino acids with hydrophobic side chains. D-aspartic acid and D-glutamic acid are not substrates for DAAO. Through the years, it has been the subject of a number of structural, functional and kinetic investigations. The most recent advances are represented by site-directed mutagenesis studies and resolution of the 3D-structure of the enzymes from pig, human and yeast. The two approaches have given us a deeper understanding of the structure-function relationships and promoted a number of investigations aimed at the modulating the protein properties. By a rational and/or a directed evolution approach, DAAO variants with altered substrate specificity (e.g., active on acidic or on all D-amino acids), increased stability (e.g., stable up to 60 degrees C), modified interaction with the flavin cofactor, and altered oligomeric state were produced. The aim of this paper is to provide an overview of the most recent research on the engineering of DAAOs to illustrate their new intriguing properties, which also have enabled us to pursue new biotechnological applications.

Assays of D-Amino Acid Oxidase Activity.

D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

Tryptophan 243 affects interprotein contacts, cofactor binding and stability in D-amino acid oxidase from Rhodotorula gracilis.

The flavoenzyme d-amino acid oxidase from Rhodotorula gracilis is a homodimeric protein whose dimeric state has been proposed to occur as a result of (a) the electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3'' of the other monomer, and (b) the interaction of residues (e.g. Trp243) belonging to the two monomers at the mixed interface region. The role of Trp243 was investigated by substituting it with either tyrosine or isoleucine: both substitutions were nondisruptive, as confirmed by the absence of significant changes in catalytic activity, but altered the tertiary structure (yielding a looser conformation) and decreased the stability towards temperature and denaturants. The change in conformation interferes both with the interaction of the coenzyme to the apoprotein moiety (although the kinetics of the apoprotein-FAD complex reconstitution process are similar between wild-type and mutant D-amino acid oxidases) and with the interaction between monomers. Our results indicate that, in the folded holoenzyme, Trp243 is situated at a position optimal for increasing the interactions between monomers by maximizing van der Waals interactions and by efficiently excluding solvent.

Regulating levels of the neuromodulator d-serine in human brain: structural insight into pLG72 and d-amino acid oxidase interaction.

The human flavoenzyme d-amino acid oxidase (hDAAO) degrades the NMDA-receptor modulator d-serine in the brain. Although hDAAO has been extensively characterized, little is known about its main modulator pLG72, a small protein encoded by the primate-specific gene G72 that has been associated with schizophrenia susceptibility. pLG72 interacts with neosynthesized hDAAO, promoting its inactivation and degradation. In this work, we used low-resolution techniques to characterize the surface topology of the hDAAO-pLG72 complex. By using limited proteolysis coupled to mass spectrometry, we could map the exposed regions in the two proteins after complex formation and highlighted an increased sensitivity to proteolysis of hDAAO in complex with pLG72. Cross-linking experiments by using bis(sulfosuccinimidyl)suberate identified the single covalent bond between T182 in hDAAO and K62 in pLG72. In order to validate the designed mode of interaction, three pLG72 variants incrementally truncated at the C terminus, in addition to a form lacking the 71 N-terminal residues, were produced. All variants were dimeric, folded, and interacted with hDAAO. The strongest decrease in affinity for hDAAO (as well as for the hydrophobic drug chlorpromazine) was apparent for the N-terminally deleted pLG72(72-153) form, which lacked K62. On the other hand, eliminating the disordered C-terminal tail yielded a more stable pLG72 protein, improved the binding to hDAAO, although giving lower enzyme inhibition. Elucidation of the mode of hDAAO-pLG72 interaction now makes it possible to design novel molecules that, by targeting the protein complex, can be therapeutically advantageous for diseases related to impairment in d-serine metabolism.

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase.

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.

Conversion of the dimeric D-amino acid oxidase from Rhodotorula gracilis to a monomeric form. A rational mutagenesis approach.

The relevance of the dimeric state for the structure/function relationships of Rhodotorula gracilis D-amino acid oxidase (RgDAAO) holoenzyme has been investigated by rational mutagenesis. Deletion of 14 amino acids in a surface loop (connecting beta-strands 12 and 13) transforms RgDAAO from a dimeric protein into a stable monomer. The mutant enzyme is still catalytically competent and retains its binding with the FAD coenzyme. Dimerization has been used by this flavoenzyme in evolution to achieve maximal activity, a tighter interaction between the protein moiety and the coenzyme, and higher thermal stability.