Activation of Phenylalanine Hydroxylase Induces Positive Cooperativity toward the Natural Cofactor

Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate l-Phenylalanine and its natural cofactor 6R-l-erythro-5,6,7,8-tetrahydrobiopterin (BH₄). Sapropterin dihydrochloride, the synthetic form of BH₄, was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH₄, a previously unknown finding. Analyses of structurally preactivated variants substantiated BH₄-dependent cooperativity of the activated enzyme that does not rely on the presence of l-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient''s metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.     

Specific Imaging of Inflammation with the 18kDa Translocator Protein Ligand DPA-714 in Animal Models of Epilepsy and Stroke

Inflammation is a pathophysiological hallmark of many diseases of the brain. Specific imaging of cells and molecules that contribute to cerebral inflammation is therefore highly desirable, both for research and in clinical application. The 18 kDa translocator protein (TSPO) has been established as a suitable target for the detection of activated microglia/macrophages. A number of novel TSPO ligands have been developed recently. Here, we evaluated the high affinity TSPO ligand DPA-714 as a marker of brain inflammation in two independent animal models. For the first time, the specificity of radiolabeled DPA-714 for activated microglia/macrophages was studied in a rat model of epilepsy (induced using Kainic acid) and in a mouse model of stroke (transient middle cerebral artery occlusion, tMCAO) using high-resolution autoradiography and immunohistochemistry. Additionally, cold-compound blocking experiments were performed and changes in blood-brain barrier (BBB) permeability were determined. Target-to-background ratios of 2 and 3 were achieved in lesioned vs. unaffected brain tissue in the epilepsy and tMCAO models, respectively. In both models, ligand uptake into the lesion corresponded well with the extent of Ox42- or Iba1-immunoreactive activated microglia/macrophages. In the epilepsy model, ligand uptake was almost completely blocked by pre-injection of DPA-714 and FEDAA1106, another high-affinity TSPO ligand. Ligand uptake was independent of the degree of BBB opening and lesion size in the stroke model. We provide further strong evidence that DPA-714 is a specific ligand to image activated microglia/macrophages in experimental models of brain inflammation.     

Unexpected wide substrate specificity of C. perfringens α-toxin phospholipase C

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the main virulence factor for gas gangrene in humans. The lipase activity serves the bacterium to generate lipid signals in the host eukaryotic cell, and ultimately to degrade the host cell membranes. Several previous reports indicated that CpPLC was specific for phosphatidylcholine and sphingomyelin. Molecular docking studies described in this paper predict favorable interactions of the CpPLC active site with other phospholipids, e.g. phosphatidylethanolamine, phosphatidylinositol and, to a lesser extent, phosphatidylglycerol. On the basis of these predictions, we have performed experimental studies showing α-toxin to degrade all the phospholipids mentioned above. The molecular docking data also provide an explanation for the observed lower activity of CpPCL on sphingomyelin as compared to the glycerophospholipids.     

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 ??mol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.     

Fetuin‐A in the developing brain

The serum protein fetuin‐A is essential for mineral homeostasis and shows immunomodulatory functions, for example by binding to TGF superfamily proteins. It proved neuroprotective in a rat stroke model and reduced lethality after systemic lipopolysaccharide challenge in mice. Serum fetuin‐A concentrations are highest during intrauterine life. Different species show intrauterine cerebral fetuin‐A immunoreactivity, suggesting a contribution to brain development. We therefore aimed at specifying fetuin‐A immunoreactivity in brains of newborn rats (age P0–P28) and human neonates (20–40 weeks of gestation). In humans and rats, fetuin‐A was found in cortex, white matter, subplate, hippocampus, subventricular zone, and ependymal cells which supports a global role for brain function. In rats, overall fetuin‐A immunoreactivity decreased with age. At P0 fetuin‐A immunoreactivity affected most brain structures. Thereafter, it became increasingly restricted to distinct cells of the hippocampus, cingular gyrus, periventricular stem cell layer, and ependyma. In ependymal cells the staining pattern complied with active transependymal transport from cerebrospinal fluid. Double immunofluorescence studies revealed colocalization with NeuN (mature neurons), beta III tubulin (immature neurons), GFAP (astrocytes), and CD68 (activated microglia). This points to a role of fetuin‐A in different brain functional systems. In human neonatal autopsy cases, frequently affected from severe neurological and non‐neurological diseases, fetuin‐A immunoreactivity was heterogeneous and much less associated with age than in healthy tissues studied earlier, suggesting an impact of exogeneous noxious factors on fetuin‐A regulation. Further research on the role of fetuin‐A in the neonatal brain during physiological and pathological conditions is recommended. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 354–369, 2013     

Habitual Functional Electrical Stimulation Therapy Improves Gait Kinematics and Walking Performance,but Not Patient-Reported Functional Outcomes,of People with Multiple Sclerosis who Present with Foot-Drop

Background

People with Multiple Sclerosis (pwMS) often experience a disturbed gait function such as foot-drop. The objective of this pilot study was to investigate the medium term effects of using Functional Electrical Stimulation (FES) to treat foot-drop over a period 12 weeks on gait and patient reported outcomes of pwMS.

Methods and Findings

Nine pwMS aged 35 to 64 (2 males, 7 females) were assessed on four occasions; four weeks before baseline, at baseline and after six weeks and twelve weeks of FES use. Joint kinematics and performance on the 10 meter and 2 minute walk tests (10WT, 2 minWT) were assessed with and without FES. Participants also completed the MS walking Scale (MSWS), MS impact scale (MSIS29), Fatigue Severity Score (FSS) and wore an activity monitor for seven days after each assessment. Compared to unassisted walking, FES resulted in statistically significant improvements in peak dorsiflexion in swing (p = 0.006), 10MWT (p = 0.006) and 2 minWT (p = 0.002). Effect sizes for the training effect, defined as the change from unassisted walking at baseline to that at 12 weeks, indicated improved ankle angle at initial contact (2.6°, 95% CI −1° to 4°, d = 0.78), and a decrease in perceived exertion over the 2 min walking tests (−1.2 points, 95% CI −5.7 to 3.4, d = −0.86). Five participants exceeded the Minimally Detectable Change (MDC) for a training effect on the 10mWT, but only two did so for the 2 minWT. No effects of the use of FES for 12 weeks were found for MSWS, MSIS29, FSS or step count.

Conclusion

Although FES to treat foot-drop appears to offer the potential for a medium term training effect on ankle kinematics and walking speed, this was not reflected in the patient reported outcomes. This observed lack of relationship between objective walking performance and patient reported outcomes warrants further investigation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01977287","term_id":"NCT01977287"}}NCT01977287     

Amide-modified prenylcysteine based Icmt inhibitors: Structure-activity relationships, kinetic analysis and cellular characterization

Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for the α-carboxyl methylation of the C-terminal isoprenylated cysteine of CaaX proteins, including Ras proteins. This specific posttranslational methylation event has been shown to be important for cellular transformation by oncogenic Ras isoforms. This finding led to interest in hIcmt inhibitors as potential anti-cancer agents. Previous analog studies based on N-acetyl-S-farnesylcysteine identified two prenylcysteine-based low micromolar inhibitors (1a and 1b) of hIcmt, each bearing a phenoxyphenyl amide modification. In this study, a focused library of analogs of 1a and 1b was synthesized and screened versus hIcmt, delineating structural features important for inhibition. Kinetic characterization of the most potent analogs 1a and 1b established that both inhibitors exhibited mixed-mode inhibition and that the competitive component predominated. Using the Cheng-Prusoff method, the K(i) values were determined from the IC(50) values. Analog 1a has a K(IC) of 1.4±0.2μM and a K(IU) of 4.8±0.5μM while 1b has a K(IC) of 0.5±0.07μM and a K(IU) of 1.9±0.2μM. Cellular evaluation of 1b revealed that it alters the subcellular localization of GFP-KRas, and also inhibits both Ras activation and Erk phosphorylation in Jurkat cells.     

Dual effect of the phorbol ester TPA on arachidonic acid release from HeLa cells

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induces release of arachidonic acid (AA) from HeLa cells with a maximum at 2-3 h. Subsequently the extracellular level of AA decreases. Cycloheximide (CH, 10(-5) M does not influence the release of AA, however, it causes the AA level to remain elevated. In the presence of TPA and CH (i) re-uptake of AA is not altered, (ii) re-incorporation of AA into phosphatidylinositol (and phosphatidylethanolamine) is largely increased, and (iii) the level of lysophosphatidylinositol is elevated. The latter two phenomena can be prevented by fluocinolone acetonide (10(-8) M), i.e. by inhibition of phospholipase A2 (PLA2). These data point to a continuously elevated PLA2 activity in the presence of TPA and CH. The phorbol ester appears to induce a proteinaceous principle which diminishes PLA2 activity.     

Allele identification using immobilized mismatch binding protein: detection and identification of antibiotic-resistant bacteria and determination of sheep susceptibility to scrapie.

A novel method for detection and identification of specific alleles has been developed utilizing immobilized mismatch binding protein (IMBP). The assay involves the use of biotin-labeled probes, which are prepared by PCR amplification of cloned fragments with known sequence. The use of probes avoids many of the problems associated with the extreme sensitivity of IMBP assays to errors in PCR amplification. The method can be used to monitor PCR fidelity and to genotype both diploid and haploid organisms and has been used to distinguish rifampicin-sensitive and -resistant strains of Mycobacterium tuberculosis and to detect and distinguish two alleles of the sheep prion protein gene involved in susceptibility to scrapie.