Meta-analysis of genome-wide association studies of gestational duration and spontaneous preterm birth identifies new maternal risk loci.

BACKGROUND: Preterm birth (<37 weeks of gestation) is a major cause of neonatal death and morbidity. Up to 40% of the variation in timing of birth results from genetic factors, mostly due to the maternal genome. METHODS: We conducted a genome-wide meta-analysis of gestational duration and spontaneous preterm birth in 68,732 and 98,370 European mothers, respectively. RESULTS: The meta-analysis detected 15 loci associated with gestational duration, and four loci associated with preterm birth. Seven of the associated loci were novel. The loci mapped to several biologically plausible genes, for example HAND2 whose expression was previously shown to decrease during gestation, associated with gestational duration, and GC (Vitamin D-binding protein), associated with preterm birth. Downstream in silico-analysis suggested regulatory roles as underlying mechanisms for the associated loci. LD score regression found birth weight measures as the most strongly correlated traits, highlighting the unique nature of spontaneous preterm birth phenotype. Tissue expression and colocalization analysis revealed reproductive tissues and immune cell types as the most relevant sites of action. CONCLUSION: We report novel genetic risk loci that associate with preterm birth or gestational duration, and reproduce findings from previous genome-wide association studies. Altogether, our findings provide new insight into the genetic background of preterm birth. Better characterization of the causal genetic mechanisms will be important to public health as it could suggest new strategies to treat and prevent preterm birth.

Elevated human placental heat shock protein 5 is associated with spontaneous preterm birth.

BACKGROUND: Specific heat shock proteins are associated with pregnancy complications, including spontaneous preterm birth (SPTB). Placental proteomics and whole exome sequencing recently suggested an association between heat shock protein HSPA5 and uncomplicated SPTB. In the present study, we investigated the localization of and possible roles for HSPA5 in SPTB. METHODS: Western blot was performed to validate the result from the previously published proteomic analysis. We used qPCR to assess mRNA expression of genes and immunohistochemistry and immunoelectron microscopy to examine localization of HSPA5 in placental tissue. We silenced the HSPA5 gene in the HTR8/SVneo human trophoblast cell line to investigate possible functions of HSPA5. RESULTS: HSPA5 was upregulated in placentas from SPTBs compared to spontaneous term births. We did not observe upregulation of HSPA5 mRNA in placental samples. The protein was localized in placental trophoblast in both spontaneous preterm and term placentas. Gene silencing of HSPA5 in human trophoblast cell culture affected the inflammatory response and decreased the expression of several proinflammatory genes. CONCLUSIONS: We suggest that upregulation of HSPA5 in the placenta is associated with spontaneous preterm labor. HSPA5 may promote the inflammatory response and alter the anti-inflammatory state of the placenta which could eventually lead to premature labor. IMPACT: We validated upregulation of HSPA5 in placentas from spontaneous preterm birth. HSPA5 was not upregulated at transcriptional level which suggests that it may be regulated post-translationally. Silencing HSPA5 in a human trophoblast-derived cell line suggested that HSPA5 promotes expression of proinflammatory cytokines. The emerging inflammation could lead to spontaneous preterm labor. Identifying inflammatory pathways and factors associated with spontaneous preterm birth increases knowledge of the molecular mechanisms of premature labor. This could provide cues to predict imminent premature labor and lead to information about how to safely maintain pregnancies.

Human placental proteomics and exon variant studies link AAT/SERPINA1 with spontaneous preterm birth.

BACKGROUND: Preterm birth is defined as live birth before 37 completed weeks of pregnancy, and it is a major problem worldwide. The molecular mechanisms that lead to onset of spontaneous preterm birth are incompletely understood. Prediction and evaluation of the risk of preterm birth is challenging as there is a lack of accurate biomarkers. In this study, our aim was to identify placental proteins that associate with spontaneous preterm birth. METHODS: We analyzed the proteomes from placentas to identify proteins that associate with both gestational age and spontaneous labor. Next, rare and potentially damaging gene variants of the identified protein candidates were sought for from our whole exome sequencing data. Further experiments we performed on placental samples and placenta-associated cells to explore the location and function of the spontaneous preterm labor-associated proteins in placentas. RESULTS: Exome sequencing data revealed rare damaging variants in SERPINA1 in families with recurrent spontaneous preterm deliveries. Protein and mRNA levels of alpha-1 antitrypsin/SERPINA1 from the maternal side of the placenta were downregulated in spontaneous preterm births. Alpha-1 antitrypsin was expressed by villous trophoblasts in the placenta, and immunoelectron microscopy showed localization in decidual fibrinoid deposits in association with specific extracellular proteins. siRNA knockdown in trophoblast-derived HTR8/SVneo cells revealed that SERPINA1 had a marked effect on regulation of the actin cytoskeleton pathway, Slit-Robo signaling, and extracellular matrix organization. CONCLUSIONS: Alpha-1 antitrypsin is a protease inhibitor. We propose that loss of the protease inhibition effects of alpha-1 antitrypsin renders structures critical to maintaining pregnancy susceptible to proteases and inflammatory activation. This may lead to spontaneous premature birth.

Integrative genetic, genomic and transcriptomic analysis of heat shock protein and nuclear hormone receptor gene associations with spontaneous preterm birth.

Heat shock proteins are involved in the response to stress including activation of the immune response. Elevated circulating heat shock proteins are associated with spontaneous preterm birth (SPTB). Intracellular heat shock proteins act as multifunctional molecular chaperones that regulate activity of nuclear hormone receptors. Since SPTB has a significant genetic predisposition, our objective was to identify genetic and transcriptomic evidence of heat shock proteins and nuclear hormone receptors that may affect risk for SPTB. We investigated all 97 genes encoding members of the heat shock protein families and all 49 genes encoding nuclear hormone receptors for their potential role in SPTB susceptibility. We used multiple genetic and genomic datasets including genome-wide association studies (GWASs), whole-exome sequencing (WES), and placental transcriptomics to identify SPTB predisposing factors from the mother, infant, and placenta. There were multiple associations of heat shock protein and nuclear hormone receptor genes with SPTB. Several orthogonal datasets supported roles for SEC63, HSPA1L, SACS, RORA, and AR in susceptibility to SPTB. We propose that suppression of specific heat shock proteins promotes maintenance of pregnancy, whereas activation of specific heat shock protein mediated signaling may disturb maternal-fetal tolerance and promote labor.

Human CPPED1 belongs to calcineurin-like metallophosphoesterase superfamily and dephosphorylates PI3K-AKT pathway component PAK4.

Protein kinases and phosphatases regulate cellular processes by reversible phosphorylation and dephosphorylation events. CPPED1 is a recently identified serine/threonine protein phosphatase that dephosphorylates AKT1 of the PI3K-AKT signalling pathway. We previously showed that CPPED1 levels are down-regulated in the human placenta during spontaneous term birth. In this study, based on sequence comparisons, we propose that CPPED1 is a member of the class III phosphodiesterase (PDE) subfamily within the calcineurin-like metallophosphoesterase (MPE) superfamily rather than a member of the phosphoprotein phosphatase (PPP) or metal-dependent protein phosphatase (PPM) protein families. We used a human proteome microarray to identify 36 proteins that putatively interact with CPPED1. Of these, GRB2, PAK4 and PIK3R2 are known to regulate the PI3K-AKT pathway. We further confirmed CPPED1 interactions with PAK4 and PIK3R2 by coimmunoprecipitation analyses. We characterized the effect of CPPED1 on phosphorylation of PAK4 and PIK3R2 in vitro by mass spectrometry. CPPED1 dephosphorylated specific serine residues in PAK4, while phosphorylation levels in PIK3R2 remained unchanged. Our findings indicate that CPPED1 may regulate PI3K-AKT pathway activity at multiple levels. Higher CPPED1 levels may inhibit PI3K-AKT pathway maintaining pregnancy. Consequences of decreased CPPED1 expression during labour remain to be elucidated.

CPPED1-targeting microRNA-371a-5p expression in human placenta associates with spontaneous delivery.

MicroRNAs (miRNAs) are important regulators of gene expression, and their expression is associated with many physiological conditions. Here, we investigated potential associations between expression levels of miRNAs in human placenta and the onset of spontaneous term birth. Using RNA sequencing, we identified 54 miRNAs differentially expressed during spontaneous term labor compared to elective term births. Expression levels of 23 miRNAs were upregulated, whereas 31 were downregulated at least 1.5-fold. The upregulated miRNA miR-371a-5p putatively targets CPPED1, expression of which decreases during spontaneous birth. We used a luciferase reporter-based assay to test whether a miR-371a-5p mimic affected translation when it bound to the 3' untranslated region of CPPED1. In this setting, the miR-371a-5p mimic resulted in lower luciferase activity, which suggests that miR-371a-5p regulates levels of CPPED1. In conclusion, inversely correlated levels of miR-371a-5p and CPPED1 suggest a role for both in spontaneous delivery.

Risk of spontaneous preterm birth and fetal growth associates with fetal SLIT2.

Spontaneous preterm birth (SPTB) is the leading cause of neonatal death and morbidity worldwide. Both maternal and fetal genetic factors likely contribute to SPTB. We performed a genome-wide association study (GWAS) on a population of Finnish origin that included 247 infants with SPTB (gestational age [GA] < 36 weeks) and 419 term controls (GA 38-41 weeks). The strongest signal came within the gene encoding slit guidance ligand 2 (SLIT2; rs116461311, minor allele frequency 0.05, p = 1.6×10-6). Pathway analysis revealed the top-ranking pathway was axon guidance, which includes SLIT2. In 172 very preterm-born infants (GA <32 weeks), rs116461311 was clearly overrepresented (odds ratio 4.06, p = 1.55×10-7). SLIT2 variants were associated with SPTB in another European population that comprised 260 very preterm infants and 9,630 controls. To gain functional insight, we used immunohistochemistry to visualize SLIT2 and its receptor ROBO1 in placentas from spontaneous preterm and term births. Both SLIT2 and ROBO1 were located in villous and decidual trophoblasts of embryonic origin. Based on qRT-PCR, the mRNA levels of SLIT2 and ROBO1 were higher in the basal plate of SPTB placentas compared to those from term or elective preterm deliveries. In addition, in spontaneous term and preterm births, placental SLIT2 expression was correlated with variations in fetal growth. Knockdown of ROBO1 in trophoblast-derived HTR8/SVneo cells by siRNA indicated that it regulate expression of several pregnancy-specific beta-1-glycoprotein (PSG) genes and genes involved in inflammation. Our results show that the fetal SLIT2 variant and both SLIT2 and ROBO1 expression in placenta and trophoblast cells may be correlated with susceptibility to SPTB. SLIT2-ROBO1 signaling was linked with regulation of genes involved in inflammation, PSG genes, decidualization and fetal growth. We propose that this receptor-ligand couple is a component of the signaling network that promotes SPTB.

Expression of CPPED1 in human trophoblasts is associated with timing of term birth.

Understanding of timing of human parturition is incomplete. Therefore, we carried out proteomic analyses of full-term placentas from uncomplicated pregnancies to identify protein signatures associated with the onset of spontaneous delivery. We found quantitative associations of 10 proteins with spontaneous term birth, evident either in the basal or in the chorionic plates or in both. Additional 18 proteins were associated according to the location within placenta indicating local variations in protein amounts. Calcineurin-like phosphoesterase domain-containing 1 (CPPED1), a phosphatase previously suggested dephosphorylating AKT1/PKB, was one of the identified proteins. qRT-PCR revealed the mRNA level of CPPED1 was higher in elective caesarean deliveries than in spontaneous births, while immunohistochemistry showed CPPED1 in cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts. Noteworthy, phosphorylation status of AKT1 did not differ between placentas from elective caesarean and spontaneous deliveries. Additionally, analyses of samples from infants indicated that single-nucleotide polymorphisms rs11643593 and rs8048866 of CPPED1 were associated with duration of term pregnancy. Finally, post-transcriptional silencing of CPPED1 in cultured HTR8/SVneo cells by siRNAs affected gene expression in pathways associated with inflammation and blood vessel development. We postulate that functions regulated by CPPED1 in trophoblasts at choriodecidual interphase have a role in the induction of term labour, but it may be independent of AKT1.

CXCR3 Polymorphism and Expression Associate with Spontaneous Preterm Birth.

Spontaneous preterm birth (SPTB) is a major factor associating with deaths and with lowered quality of life in humans. Environmental and genetic factors influence the susceptibility. Previously, by analyzing families with recurrent SPTB in linkage analysis, we identified a linkage peak close to the gene encoding CXCR3. Present objectives were to investigate the association of CXCR3 with SPTB in Finnish mothers (n = 443) and infants (n = 747), to analyze CXCR3 expression levels in human placenta and levels of its ligands in umbilical cord blood, and to verify the influence of Cxcr3 on SPTB-associating cytokines in mice. We detected an association between an intronic CXCR3 polymorphism, rs2280964, and SPTB in infants from families with recurrent preterm births (p = 0.009 versus term controls, odds ratio 0.52, 95% confidence interval 0.32-0.86). The minor allele was protective and undertransmitted to SPTB infants (p = 0.007). In the placenta and fetal membranes, the rs2280964 major allele homozygotes had higher expression levels than minor allele homozygotes; decidual trophoblasts showed strong CXCR3 immunoreactivity. Expression was higher in SPTB placentas compared with those from elective deliveries. Concentration of a CXCR3 ligand, CXCL9, was increased in cord blood from SPTB, and the protective rs2280964 allele was associated with low CXCL9. In CXCR3-deficient mice (Mus musculus), SPTB-associating cytokines were not acutely increased in amniotic fluid after preterm birth-inducing dose of maternal LPS. Our results indicate that CXCR3 contributes to SPTB. Activation of CXCR3 signaling may disturb the maternal-fetal tolerance, and this may promote labor.

Salmonella enterica MTAN at 1.36 Å resolution: a structure-based design of tailored transition state analogs.

Accumulation of 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) in bacteria disrupts the S-adenosylmethionine pool to alter biological methylations, synthesis of polyamines, and production of quorum-sensing molecules. Bacterial metabolism of MTA and SAH depends on MTA/SAH nucleosidase (MTAN), an enzyme not present in humans and a target for quorum sensing because MTAN activity is essential for synthesis of autoinducer-2 molecules. Crystals of Salmonella enterica MTAN with product and transition state analogs of MTA and SAH explain the structural contacts causing pM binding affinity for the inhibitor and reveal a "water-wire" channel for the catalytic nucleophile. The crystal structure shows an extension of the binding pocket filled with polyethylene glycol. We exploited this discovery by the design and synthesis of tailored modifications of the currently existing transition state analogs to fill this site. This site was not anticipated in MTAN structures. Tailored inhibitors with dissociation constants of 5 to 15 pM are characterized.

Catalytic site conformations in human PNP by 19F-NMR and crystallography.

Purine nucleoside phosphorylase (PNP) is a target for leukemia, gout, and autoimmune disorders. Dynamic motion of catalytic site loops has been implicated in catalysis, but experimental evidence was lacking. We replaced catalytic site groups His257 or His64 with 6-fluoro-tryptophan (6FW) as site-specific NMR probes. Conformational adjustments in the 6FW-His257-helical and His64-6FW-loop regions were characterized in PNP phosphate-bound enzyme and in complexes with catalytic site ligands, including transition state analogs. Chemical shift and line-shape changes associated with these complexes revealed dynamic coexistence of several conformational states in these regions in phosphate-bound enzyme and altered or single conformations in other complexes. These conformations were also characterized by X-ray crystallography. Specific (19)F-Trp labels and X-ray crystallography provide multidimensional characterization of conformational states for free, catalytic, and inhibited complexes of human PNP.

A picomolar transition state analogue inhibitor of MTAN as a specific antibiotic for Helicobacter pylori.

Campylobacter and Helicobacter species express a 6-amino-6-deoxyfutalosine N-ribosylhydrolase (HpMTAN) proposed to function in menaquinone synthesis. BuT-DADMe-ImmA is a 36 pM transition state analogue of HpMTAN, and the crystal structure of the enzyme-inhibitor complex reveals the mechanism of inhibition. BuT-DADMe-ImmA has a MIC(90) value of <8 ng/mL for Helicobacter pylori growth but does not cause growth arrest in other common clinical pathogens, thus demonstrating potential as an H. pylori-specific antibiotic.

Femtomolar inhibitors bind to 5'-methylthioadenosine nucleosidases with favorable enthalpy and entropy.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolytic cleavage of adenine from methylthioadenosine (MTA). Inhibitor design and synthesis informed by transition state analysis have developed femtomolar inhibitors for MTANs, among the most powerful known noncovalent enzyme inhibitors. Thermodynamic analyses of the inhibitor binding reveals a combination of highly favorable contributions from enthalpic (-24.7 to -4.0 kcal mol(-1)) and entropic (-10.0 to 6.4 kcal mol(-1)) interactions. Inhibitor binding to similar MTANs from different bacterial species gave distinct energetic contributions from similar catalytic sites. Thus, binding of four transition state analogues to EcMTAN and SeMTAN is driven primarily by enthalpy, while binding to VcMTAN is driven primarily by entropy. Human MTA phosphorylase (hMTAP) has a transition state structure closely related to that of the bacterial MTANs, and it binds tightly to some of the same transition state analogues. However, the thermodynamic signature of binding of an inhibitor to hMTAP differs completely from that with MTANs. We conclude that factors other than first-sphere catalytic residue contacts contribute to binding of inhibitors because the thermodynamic signature differs between bacterial species of the same enzyme.

Three Japanese Patients with Beta-Ketothiolase Deficiency Who Share a Mutation, c.431A>C (H144P) in ACAT1 : Subtle Abnormality in Urinary Organic Acid Analysis and Blood Acylcarnitine Analysis Using Tandem Mass Spectrometry.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency affects both isoleucine catabolism and ketone body metabolism. The disorder is characterized by intermittent ketoacidotic episodes. We report three Japanese patients. One patient (GK69) experienced two ketoacidotic episodes at the age of 9 months and 3 years, and no further episodes until the age of 25 years. She had two uncomplicated pregnancies. GK69 was a compound heterozygote of the c.431A>C (H144P) and c.1168T>C (S390P) mutations in T2 (ACAT1) gene. She was not suspected of having T2 deficiency during her childhood, but she was diagnosed as T2 deficient at the age of 25 years by enzyme assay using fibroblasts. The other two patients were identical twin siblings who presented their first ketoacidotic crisis simultaneously at the age of 3 years 4 months. One of them (GK77b) died during the first crisis and the other (GK77) survived. Even during severe crises, C5-OH and C5:1 were within normal ranges in their blood acylcarnitine profiles and trace amounts of tiglylglycine and small amounts of 2-methyl-3-hydroxybutyrate were detected in their urinary organic acid profiles. They were H144P homozygotes. This H144P mutation has retained the highest residual T2 activity in the transient expression analysis of mutant cDNA thus far, while the S390P mutation did not retain any residual T2 activity. The "mild" H144P mutation may result in subtle profiles in blood acylcarnitine and urinary organic acid analyses. T2-deficient patients with "mild" mutations have severe ketoacidotic crises but their chemical phenotypes may be subtle even during acute crises.

The characterization and evolutionary relationships of a trypanosomal thiolase.

Thiolases are enzymes that remove an acetyl-coenzyme A group from acyl-CoA in the catabolic ß-oxidation of fatty acids, or catalyse the reverse condensation reaction for anabolic processes such as the biosynthesis of sterols and ketone bodies. In humans, six homologous isoforms of thiolase have been described, differing from each other in sequence, oligomeric state, substrate specificity and subcellular localization. A bioinformatics analysis of parasite genomes, being (i) different species of African trypanosomes, (ii) Trypanosoma cruzi and (iii) Leishmania spp., using the six human sequences as queries, showed that the distribution of thiolases in human and each of the studied Trypanosomatidae is completely different. Only one of these isoforms, called SCP2-thiolase, was found in each of the Trypanosomatidae, whereas the TFE-thiolase was also found in T. cruzi and Leishmania spp., and the AB-thiolase only in T. cruzi. Each of the trypanosomatid thiolases clusters with its orthologues from other organisms in a phylogenetic analysis and shares with them the isoform-specific sequence fingerprints. The single T. brucei SCP2-thiolase has been expressed in Escherichia coli and characterized. It shows activity in both the degradative and synthetic directions. Transcripts of this thiolase were detected in both bloodstream- and procyclic-form trypanosomes, but the protein was found only in the procyclic form. The encoded protein has both a predicted N-terminal mitochondrial signal peptide and a C-terminal candidate type 1 peroxisomal-targeting signal for sorting it into glycosomes. However experimentally, only a mitochondrial localization was found for both procyclic trypanosomes grown with glucose and cells cultured with amino acids as an energy source. When the thiolase expression in procyclic cells was knocked down by RNA interference, no important change in growth rate occurred, irrespective of whether the cells were grown with or without glucose, indicating that the metabolic pathway(s) involving this enzyme is/are not essential for the parasite under either of these growth conditions.

Mitochondrial fatty acid synthesis--an adopted set of enzymes making a pathway of major importance for the cellular metabolism.

The highly conserved fatty acid de novo synthesis pathway in mitochondria proceeds in an acyl carrier protein-dependent manner through a discrete set of enzymes. Saccharomyces cerevisiae has served as a model for studies of mitochondrial fatty acid synthesis type II (FAS II) and as a template for identification of mammalian components. Inactivation of mitochondrial FAS II in yeast results in respiratory deficiency and loss of cytochromes. The pathway produces the octanoyl-ACP substrate for lipoic acid synthesis, but several pieces of evidence indicate that it is capable of the generation of longer fatty acids. A number of structures of mitochondrial FAS II enzymes have been published in the past few years, allowing for a comparison with their prokaryotic counterparts, several of which have been described as promising targets for antibiotics. Recently, novel links between mitochondrial FAS and RNA processing in yeast and vertebrates have been reported. In S. cerevisiae, deficiency in mitochondrial FAS results in failure of maturation of mitochondrial RNAse P, while, in mammals, mitochondrial 3-hydroxyacyl thioester dehydratase and the RPP14 subunit of RNase P are encoded by the same bicistronic transcript. The first publications linking mitochondrial FAS II to disease states in mammals are emerging.

Alternative conformations of the x region of human protein disulphide-isomerase modulate exposure of the substrate binding b' domain.

Protein disulphide isomerase (PDI) is a key multi-domain protein folding catalyst in the endoplasmic reticulum. The b' domain of PDI is essential for the non-covalent binding of incompletely folded protein substrates. Earlier, we defined the substrate binding site in the b' domain of human PDI by modelling and mutagenesis studies. Here, we show by fluorescence and NMR that recombinant human PDI b'x (comprising the b' domain and the subsequent x linker region) can assume at least two different conformations in solution. We have screened mutants in the b'x region to identify mutations that favour one of these conformers in recombinant b'x, and isolated and characterised examples of both types. We have crystallised one mutant of b'x (I272A mutation) in which one conformer is stabilized, and determined its crystal structure to a resolution of 2.2 A. This structure shows that the b' domain has the typical thioredoxin fold and that the x region can interact with the b' domain by "capping" a hydrophobic site on the b' domain. This site is most likely the substrate binding site and hence such capping will inhibit substrate binding. All of the mutations we previously reported to inhibit substrate binding shift the equilibrium towards the capped conformer. Hence, these mutations act by altering the natural equilibrium and decreasing the accessibility of the substrate binding site. Furthermore, we have confirmed that the corresponding structural transition occurs in the wild type full-length PDI. A cross-comparison of our data with that for other PDI-family members, Pdi1p and ERp44, suggests that the x region of PDI can adopt alternative conformations during the functional cycle of PDI action and that these are linked to the ability of PDI to interact with folding substrates.

Structural enzymological studies of 2-enoyl thioester reductase of the human mitochondrial FAS II pathway: new insights into its substrate recognition properties.

Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway.

Crystallographic and kinetic studies of human mitochondrial acetoacetyl-CoA thiolase: the importance of potassium and chloride ions for its structure and function.

Thiolases are CoA-dependent enzymes which catalyze the formation of a carbon-carbon bond in a Claisen condensation step and its reverse reaction via a thiolytic degradation mechanism. Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as for the degradation of 2-methylacetoacetyl-CoA. Human T2 deficiency has been identified in more than 60 patients. A unique property of T2 is its activation by potassium ions. High-resolution human T2 crystal structures are reported for the apo form and the CoA complex, with and without a bound potassium ion. The potassium ion is bound near the CoA binding site and the catalytic site. Binding of the potassium ion at this low-affinity binding site causes the rigidification of a CoA binding loop and an active site loop. Unexpectedly, a high-affinity binding site for a chloride ion has also been identified. The chloride ion is copurified, and its binding site is at the dimer interface, near two catalytic loops. A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate, whereas the other structurally characterized thiolases cannot utilize the 2-methylated compounds. The kinetic measurements show that T2 can degrade acetoacetyl-CoA and 2-methylacetoacetyl-CoA with similar catalytic efficiencies. For both substrates, the turnover numbers increase approximately 3-fold when the potassium ion concentration is increased from 0 to 40 mM KCl. The structural analysis of the active site of T2 indicates that the Phe325-Pro326 dipeptide near the catalytic cavity is responsible for the exclusive 2-methyl-branched substrate specificity.

Kinetic and expression analyses of seven novel mutations in mitochondrial acetoacetyl-CoA thiolase (T2): identification of a Km mutant and an analysis of the mutational sites in the structure.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects isoleucine catabolism and ketone body metabolism. We identified 7 novel and 2 previously reported mutations in six T2-deficient patients. Transient expression analysis of wild-type and eight mutant cDNAs was performed at 40, 37 and 30 degrees C. Although no significant residual activity was detected, mutant proteins were detected in the N158D, N158S, R208Q, Y219H and N282H mutants. Accumulation of these mutant proteins was temperature-sensitive with the highest expression levels at lower temperatures. Expression of Q73P and N353K cDNAs yielded neither residual T2 protein nor enzyme activity. An E252del mutant T2 was detected with a relative protein amount and enzyme activity of 30% and 25%, respectively, in comparison to the wild-type at 37 degrees C. The E252del mutant protein was more stable at 30 degrees C expression than 37 degrees C, but was essentially undetectable at 40 degrees C, indicating its temperature-sensitive instability. Kinetic studies revealed a twofold K(m) elevation for substrates coenzyme A and acetoacetyl-CoA in the E252del mutant, while V(max) was comparable to the wild-type. We conclude that the E252del is a temperature-sensitive K(m) mutant. This correlates well with the effect predicted from the T2 tertiary structure analysis, using the crystal structure of the human T2 homotetramer. The probable effect of the other mutations on the T2 tertiary structure was also evaluated.