Structural aspects of HDAC8 mechanism and dysfunction in Cornelia de Lange syndrome spectrum disorders

Cornelia de Lange Syndrome (CdLS) encompasses a broad spectrum of phenotypes characterized by distinctive craniofacial abnormalities, limb malformations, growth retardation, and intellectual disability. CdLS spectrum disorders are referred to as cohesinopathies, with ～70% of patients having a mutation in a gene encoding a core cohesin protein (SMC1A, SMC3, or RAD21) or a cohesin regulatory protein (NIPBL or HDAC8). Notably, the regulatory function of HDAC8 in cohesin biology has only recently been discovered. This Zn²⁺‐dependent hydrolase catalyzes the deacetylation of SMC3, a necessary step for cohesin recycling during the cell cycle. To date, 23 different missense mutants in the gene encoding HDAC8 have been identified in children with developmental features that overlap those of CdLS. Enzymological, biophysical, and structural studies of CdLS HDAC8 protein mutants have yielded critical insight on compromised catalysis in vitro. Most CdLS HDAC8 mutations trigger structural changes that directly or indirectly impact substrate binding and catalysis. Additionally, several mutations significantly compromise protein thermostability. Intriguingly, catalytic activity in many HDAC8 mutants can be partially or fully restored by an N‐acylthiourea activator, suggesting a plausible strategy for the chemical rescue of compromised HDAC8 catalysis in vivo.     

On the Molecular Basis of D-Bifunctional Protein Deficiency Type III

Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K_m, V_max and k_cat) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.     

Choline Acetyltransferase-Deficient Mutants of the Nematode CAENORHABDITIS ELEGANS

We have identified five independent allelic mutations, defining the gene cha-1, that result in decreased choline acetyltransferase (ChAT) activity in Caenorhabditis elegans. Four of the mutant alleles, when homozygous, lead to ChAT reductions of>98%, as well as recessive phenotypes of uncoordinated behavior, small size, slow growth and resistance to cholinesterase inhibitors. Animals homozygous for the fifth allele retain approximately 10% of the wild-type enzyme level; purified enzyme from this mutant has altered K_m values for both choline and acetyl-CoA and is more thermolabile than the wild-type enzyme. These qualitative alterations, together with gene dosage data, argue that cha-1 is the structural gene for ChAT. cha-1 has been mapped to the left arm of linkage group IV and is within 0.02 map unit of the gene unc-17, mutant alleles of which lead to all of the phenotypes of cha-1 mutants except for the ChAT deficiency. Extensive complementation studies of cha-1 and unc-17 alleles reveal a complex complementation pattern, suggesting that both loci may be part of a single complex gene.     

The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus: THE ROLE OF GLU232 IN CATALYSIS*

The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu²³² in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that k_red, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu²³² being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both k_red and k_red/K_d from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu²³² of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pK_a of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.     

Molecular modelling and dynamics of CA2 missense mutations causative to carbonic anhydrase 2 deficiency syndrome

Abstract

Carbonic anhydrase 2 (CA2) enzyme deficiency caused by CA2 gene mutations is an inherited disorder characterized by symptoms like osteopetrosis, renal tubular acidosis, and cerebral calcification. This study has collected the CA2 deficiency causal missense mutations and assessed their pathogenicity using diverse computational programs. The 3D protein models for all missense mutations were built, and analyzed for structural divergence, protein stability, and molecular dynamics properties. We found M-CAP as the most sensitive prediction method to measure the deleterious potential of CA2 missense mutations. Free energy dynamics of tertiary structure models of CA2 mutants with DUET, mCSM, and SDM based consensus methods predicted only 50% of the variants as destabilizing. Superimposition of native and mutant CA2 models revealed the minor structural fluctuations at the amino acid residue level but not at the whole protein structure level. Near native molecular dynamic simulation analysis indicated that CA2 causative missense variants result in residue level fluctuation pattern in the protein structure. This study expands the understanding of genotype-protein phenotype correlations underlying CA2 variant pathogenicity and presents a potential avenue for modifying the CA2 deficiency by targeting biophysical structural features of CA2 protein.

Communicated by Ramaswamy H. Sarma     

The genetics and biochemistry of urease in Ustilago violacea

Two complementing loci in different linkage groups of the basidiomycete Ustilago violacea are involved in urease activity: a structural one (ure-1) and a second inferred to involve a permease (ure-2) locus. Two types of complementing mutations occur in the structural locus: null activity (ure-1a) and obviously reduced activity (ure-1b). The ure-2 mutants lacked urease activity in vivo on the phenol red-urea test medium, but gave extracts with wild-type activity. Extracts from wild-type strains gave one site of urease activity after polyacrylamide gel electrophoresis. A number of ure-1b mutants and active revertants from ure-1a mutants yielded electrophoretically variant urease sites. The results are discussed in terms of enzyme polymorphism in haploid eukaryotes by one (missense) or two (null, then missense) mutations.     

Virtual screening of mandelate racemase mutants with enhanced activity based on binding energy in the transition state

Mandelate racemase (MR) is a promising candidate for the dynamic kinetic resolution of racemates. However, the poor activity of MR towards most of its non-natural substrates limits its widespread application. In this work, a virtual screening method based on the binding energy in the transition state was established to assist in the screening of MR mutants with enhanced catalytic efficiency. Using R-3-chloromandelic acid as a model substrate, a total of 53 mutants were constructed based on rational design in the two rounds of screening. The number of mutants for experimental validation was brought down to 17 by the virtual screening method, among which 14 variants turned out to possess improved catalytic efficiency. The variant V26I/Y54V showed 5.2-fold higher catalytic efficiency (k_cat/K_m) towards R-3-chloromandelic acid than that observed for the wild-type enzyme. Using this strategy, mutants were successfully obtained for two other substrates, R-mandelamide and R-2-naphthylglycolate (V26I and V29L, respectively), both with a 2-fold improvement in catalytic efficiency. These results demonstrated that this method could effectively predict the trend of mutational effects on catalysis. Analysis from the energetic and structural assays indicated that the enhanced interactions between the active sites and the substrate in the transition state led to improved catalytic efficiency. It was concluded that this virtual screening method based on the binding energy in the transition state was beneficial in enzyme rational redesign and helped to better understand the catalytic properties of the enzyme.     

Ala258Phe substitution in Bacillus sp. YX-1 glucose dehydrogenase improves its substrate preference for xylose

Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg⁻¹ and nearly 8-folds higher k_cat/K_m value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.     

Herbicide Resistance in Datura innoxia: Kinetic Characterization of Acetolactate Synthase from Wild-Type and Sulfonylurea-Resistant Cell Variants

Acetolactate synthase (ALS, EC 4. 1.3. 18), the first enzyme in the biosynthesis of branched-chain amino acids, was isolated from wild-type and sulfonylurea-resistant Datura innoxia cell variants and characterized. Apparent K_m values of the ALS for pyruvate from three sulfonylurea-resistant variants (CSR2, CSR6, and CSR10) were manyfold greater than that of the wild type. The inhibition of wild-type and herbicide-resistant ALS activity by chlorsulfuron (CS), a sulfonylurea herbicide, and l-leucine (l-Leu), one of the feedback inhibitors of the enzyme, was examined. ALS from two CS-resistant variants exhibited severalfold greater resistance to CS than did the wild-type enzyme. Inhibition of ALS by l-Leu fitted a partially competitive pattern most closely. It is proposed that the herbicide resistance mutation accentuated the partial inhibition characteristics of ALS by l-Leu. ALS from one of the two CS-resistant variants (CSR6) had a K_i for l-Leu an order of magnitude greater than that of the wild-type enzyme. The alterations in kinetic properties observed in the ALS from sulfonylurea-resistant variants are discussed in relation to the possible evolutionary significance of the herbicide binding site of this enzyme, the physiological effects of such biochemical alterations, and their practical utility in genetic studies.     

In Vivo Analysis of the Major Exocytosis-sensitive Phosphoprotein in Tetrahymena

Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, ΔPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.     

Probing the role of a conserved salt bridge in the intramolecular electron transfer kinetics of human sulfite oxidase

Sulfite oxidase (SO) is a vital metabolic enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed mechanism of this molybdenum cofactor dependent enzyme involves two one-electron intramolecular electron transfer (IET) steps from the molybdenum center to the iron of the b ₅-type heme and two one-electron intermolecular electron transfer steps from the heme to cytochrome c. This work focuses on how the electrostatic interaction between two conserved amino acid residues, R472 and D342, in human SO (hSO) affects catalysis. The hSO variants R472M, R472Q, R472K, R472D, and D342K were created to probe the effect of the position of the salt bridge charges, along with the interaction between these two residues. With the exception of R472K, these variants all showed a significant decrease in their IET rate constants, k _et, relative to wild-type hSO, indicating that the salt bridge between residues 472 and 342 is important for rapid IET. Surprisingly, however, except for R472K and R472D, all of the variants show k _cat values higher than their corresponding k _et values. The turnover number for R472D is about the same as k _et, which suggests that the change in this variant is rate-limiting in catalysis. Direct spectroelectrochemical determination of the Fe(III/II) reduction potentials of the heme and calculation of the Mo(VI/V) potentials revealed that all of the variants affected the redox potentials of both metal centers, probably due to changes in their environments. Thus, the position of the positive charge of R472 and that of the negative charge of D342 are both important in hSO, and changing either the position or the nature of these charges perturbs IET and catalysis.     

High tolerance to mutations in a Chlamydia trachomatis peptide deformylase loop

AIM: To determine if and how a loop region in the peptide deformylase (PDF) of Chlamydia trachomatis regulates enzyme function.METHODS: Molecular dynamics simulation was used to study a structural model of the chlamydial PDF (cPDF) and predict the temperature factor per residue for the protein backbone atoms. Site-directed mutagenesis was performed to construct cPDF variants. Catalytic properties of the resulting variants were determined by an enzyme assay using formyl-Met-Ala-Ser as a substrate.RESULTS: In silico analysis predicted a significant increase in atomic motion in the DGELV sequence (residues 68-72) of a loop region in a cPDF mutant, which is resistant to PDF inhibitors due to two amino acid substitutions near the active site, as compared to wild-type cPDF. The D68R and D68R/E70R cPDF variants demonstrated significantly increased catalytic efficiency. The E70R mutant showed only slightly decreased efficiency. Although deletion of residues 68-72 resulted in a nearly threefold loss in substrate binding, this deficiency was compensated for by increased catalytic efficiency.CONCLUSION: Movement of the DGELV loop region is involved in a rate-limiting conformational change of the enzyme during catalysis. However, there is no stringent sequence requirement for this region for cPDF enzyme activity.     

Structure-function analysis of Friedreich's ataxia mutants reveals determinants of frataxin binding and activation of the Fe-S assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k(cat)/K(M) higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k(cat)/K(M) of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.     

Genetic interactions between planar cell polarity genes cause diverse neural tube defects in mice

Neural tube defects (NTDs) are among the commonest and most severe forms of developmental defect, characterized by disruption of the early embryonic events of central nervous system formation. NTDs have long been known to exhibit a strong genetic dependence, yet the identity of the genetic determinants remains largely undiscovered. Initiation of neural tube closure is disrupted in mice homozygous for mutations in planar cell polarity (PCP) pathway genes, providing a strong link between NTDs and PCP signaling. Recently, missense gene variants have been identified in PCP genes in humans with NTDs, although the range of phenotypes is greater than in the mouse mutants. In addition, the sequence variants detected in affected humans are heterozygous, and can often be detected in unaffected individuals. It has been suggested that interactions between multiple heterozygous gene mutations cause the NTDs in humans. To determine the phenotypes produced in double heterozygotes, we bred mice with all three pairwise combinations of Vangl2^Lp, Scrib^Crc and Celsr1^Crsh mutations, the most intensively studied PCP mutants. The majority of double-mutant embryos had open NTDs, with the range of phenotypes including anencephaly and spina bifida, therefore reflecting the defects observed in humans. Strikingly, even on a uniform genetic background, variability in the penetrance and severity of the mutant phenotypes was observed between the different double-heterozygote combinations. Phenotypically, Celsr1^Crsh;Vangl2^Lp;Scrib^Crc triply heterozygous mutants were no more severe than doubly heterozygous or singly homozygous mutants. We propose that some of the variation between double-mutant phenotypes could be attributed to the nature of the protein disruption in each allele: whereas Scrib^Crc is a null mutant and produces no Scrib protein, Celsr1^Crsh and Vangl2^Lp homozygotes both express mutant proteins, consistent with dominant effects. The variable outcomes of these genetic interactions are of direct relevance to human patients and emphasize the importance of performing comprehensive genetic screens in humans.KEY WORDS: Neural tube defects, Planar cell polarity, Genetic interactions, Craniorachischisis, Multiple heterozygosity     

LcrV Mutants That Abolish Yersinia Type III Injectisome Function

LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR⁺] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV_*327). The addition of at least four amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR⁻ phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, LcrV_*327 mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with either injectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR⁺, LCR⁻, or dominant negative LCR⁻ phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor.     

Dysfunction of the Scn8a Voltage-gated Sodium Channel Alters Sleep Architecture,Reduces Diurnal Corticosterone Levels,and Enhances Spatial Memory

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of transient depolarizing currents and play a critical role in the electrical signaling between neurons. A null mutation in the VGSC gene SCN8A, which encodes the transmembrane protein Na_v1.6, was identified previously in a human family. Heterozygous mutation carriers displayed a range of phenotypes, including ataxia, cognitive deficits, and emotional instability. A possible role for SCN8A was also proposed in studies examining the genetic basis of attempted suicide and bipolar disorder. In addition, mice with a Scn8a loss-of-function mutation (Scn8a^med-Tg/+) show altered anxiety and depression-like phenotypes. Because psychiatric abnormalities are often associated with altered sleep and hormonal patterns, we evaluated heterozygous Scn8a^med-jo/+ mutants for alterations in sleep-wake architecture, diurnal corticosterone levels, and behavior. Compared with their wild-type littermates, Scn8a^med-jo/+ mutants experience more non-rapid eye movement (non-REM) sleep, a chronic impairment of REM sleep generation and quantity, and a lowered and flattened diurnal rhythm of corticosterone levels. No robust differences were observed between mutants and wild-type littermates in locomotor activity or in behavioral paradigms that evaluate anxiety or depression-like phenotypes; however, Scn8a^med-jo/+ mutants did show enhanced spatial memory. This study extends the spectrum of phenotypes associated with mutations in Scn8a and suggests a novel role for altered sodium channel function in human sleep disorders.     

Carbamoyl phosphate synthetase 1 deficiency in Italy: clinical and genetic findings in a heterogeneous cohort

Carbamoyl Phosphate Synthetase 1 deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder, potentially leading to lethal hyperammonemia. Based on the age of onset, there are two distinct phenotypes: neonatal and late form. The CPS1 enzyme, located in the mitochondrial matrix of hepatocytes and epithelial cells of intestinal mucosa, is encoded by the CPS1 gene. At present more than 220 clear-cut genetic lesions leading to CPS1D have been reported. As most of them are private mutations diagnosis is complicated.Here we report an overview of the main clinical findings and biochemical and molecular data of 13 CPS1D Italian patients. In two of them, one with the neonatal form and one with the late form, cadaveric auxiliary liver transplant was performed. Mutation analysis in these patients identified 17 genetic lesions, 9 of which were new confirming their “private” nature. Seven of the newly identified mutations were missense/nonsense changes. In order to study their protein level effects, we performed an in silico analysis whose results indicate that the amino acid substitutions occur at evolutionary conserved positions and affect residues necessary for enzyme stability or function.