411 research outputs found
Calmodulins from Schistosoma mansoni: Biochemical analysis and interaction with IQ-motifs from voltage-gated calcium channels.
The trematode Schistosoma mansoni is a causative agent of schistosomiasis, the second most common parasitic disease of humans after malaria. Calcium homeostasis and calcium-mediated signalling pathways are of particular interest in this species. The drug of choice for treating schistosomiasis, praziquantel, disrupts the regulation of calcium uptake and there is interest in exploiting calcium-mediated processes for future drug discovery. Calmodulin is a calcium sensing protein, present in most eukaryotes. It is a critical regulator of processes as diverse as muscle contraction, cell division and, partly through interaction with voltage-gated calcium channels, intra-cellular calcium concentrations. S. mansoni expresses two highly similar calmodulins – SmCaM1 and SmCaM2. Both proteins interact with calcium, manganese, cadmium (II), iron (II) and lead ions in native gel electrophoresis. These ions also cause conformational changes in the proteins resulting in the exposure of a more hydrophobic surface (as demonstrated by anilinonaphthalene-8-sulfonate fluorescence assays). The proteins are primarily dimeric in the absence of calcium ions, but monomeric in the presence of this ion. Both SmCaM1 and SmCaM2 interact with a peptide corresponding to an IQ-motif derived from the α-subunit of the voltage-gated calcium channel SmCav1B (residues 1923-1945). Both proteins bound with slightly higher affinity in the presence of calcium ions. However, there was no difference between the affinities of the two proteins for the peptide. This interaction could be antagonised by chlorpromazine and trifluoperazine, but not praziquantel or thiamylal. Interestingly no interaction could be detected with the other three IQ- motifs identified in S. mansoni voltage-gated ion calcium channels
Insight into the mechanism of galactokinase: role of a critical glutamate residue and helix/coil transitions
Galactokinase, the enzyme which catalyses the first committed step in the Leloir pathway, has attracted interest due to its potential as a biocatalyst and as a possible drug target in the treatment of type I galactosemia. The mechanism of the enzyme is not fully elucidated. Molecular dynamics (MD) simulations of galactokinase with the active site residues Arg-37 and Asp-186 altered predicted that two regions (residues 174-179 and 231-240) had different dynamics as a consequence. Interestingly, the same two regions were also affected by alterations in Arg-105, Glu-174 and Arg- 228. These three residues were identified as important in catalysis in previous computational studies on human galactokinase. Alteration of Arg-105 to methionine resulted in a modest reduction in activity with little change in stability. When Arg-228 was changed to methionine, the enzyme’s interaction with both ATP and galactose was affected. This variant was significantly less stable than the wild-type protein. Changing Glu-174 to glutamine (but not to aspartate) resulted in no detectable activity and a less stable enzyme. Overall, these combined in silico and in vitro studies demonstrate the importance of a negative charge at position 174 and highlight the critical role of the dynamics in to key regions of the protein. We postulate that these regions may be critical for mediating the enzyme’s structure and function.
Galactosemia: Towards Pharmacological Chaperones
Galactosemia is a rare inherited metabolic disease resulting from mutations in the four
genes which encode enzymes involved in the metabolism of galactose. The current therapy, the
removal of galactose from the diet, is inadequate. Consequently, many patients suffer lifelong
physical and cognitive disability. The phenotype varies from almost asymptomatic to life-threatening
disability. The fundamental biochemical cause of the disease is a decrease in enzymatic activity due
to failure of the affected protein to fold and/or function correctly. Many novel therapies have been
proposed for the treatment of galactosemia. Often, these are designed to treat the symptoms and
not the fundamental cause. Pharmacological chaperones (PC) (small molecules which correct the
folding of misfolded proteins) represent an exciting potential therapy for galactosemia. In theory,
they would restore enzyme function, thus preventing downstream pathological consequences. In
practice, no PCs have been identified for potential application in galactosemia. Here, we review the
biochemical basis of the disease, identify opportunities for the application of PCs and describe how
these might be discovered. We will conclude by considering some of the clinical issues which will
affect the future use of PCs in the treatment of galactosemia.ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency
RTI2018-096246-B-I00FEDER/Junta de AndalucÃa - ConsejerÃa de Transformación Económica, Industria, Conocimiento y Universidades
P18-RT-241
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