Molecular dynamics and protein frustration analysis of human fused in Sarcoma protein variants in Amyotrophic Lateral Sclerosis type 6: An In Silico approach.

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disorder. The disease is characterized by degeneration of upper and lower motor neurons, leading to death usually within five years after the onset of symptoms. While most cases are sporadic, 5%-10% of cases can be associated with familial inheritance, including ALS type 6, which is associated with mutations in the Fused in Sarcoma (FUS) gene. This work aimed to evaluate how the most frequent ALS-related mutations in FUS, R521C, R521H, and P525L affect the protein structure and function. We used prediction algorithms to analyze the effects of the non-synonymous single nucleotide polymorphisms and performed evolutionary conservation analysis, protein frustration analysis, and molecular dynamics simulations. Most of the prediction algorithms classified the three mutations as deleterious. All three mutations were predicted to reduce protein stability, especially the mutation R521C, which was also predicted to increase chaperone binding tendency. The protein frustration analysis showed an increase in frustration in the interactions involving the mutated residue 521C. Evolutionary conservation analysis showed that residues 521 and 525 of human FUS are highly conserved sites. The molecular dynamics results indicate that protein stability could be compromised in all three mutations. They also affected the exposed surface area and protein compactness. The analyzed mutations also displayed high flexibility in most residues in all variants, most notably in the interaction site with the nuclear import protein of FUS.

In silico analysis and molecular dynamics simulation of human superoxide dismutase 3 (SOD3) genetic variants.

Oxidative stress is a major factor in aging processes. Superoxide dismutase 3 (SOD3) plays a key role in the protection of extracellular oxidative stress. Missense mutations in SOD3 have been described to be associated with the occurrence of pulmonary, cardiovascular, and neoplastic diseases. This study aims to analyze the effects of missense mutations on the SOD3 structure and function by modeling a complete SOD3 structure as well as analyzing the differences between the wild-types and mutants using computational simulations. Here, ten algorithms were used to predict the structural and functional effects of missense mutations. A complete model of SOD3 protein was made by ab initio and comparative modeling using the Rosetta algorithm and validated by PROCHECK, Verify 3D, QMEAN, and ProSa. Molecular dynamics (MD) simulations were performed and analyzed using the GROMACS package. The deleterious potential of the A58T and R231G mutants was not predicted by the majority of the used algorithms. The analyzed mutations were predicted as destabilizing by at least one algorithm. The MD analyses indicated that protein flexibility may be increased by all of the analyzed mutations, while the protein-ligand stability may be decreased. They also suggested that the variants A91T and R231G increase the overall dimensions of SOD3 and decrease its accessible surface area. Our findings, therefore, indicated that the analyzed mutations could affect the protein structure and its ability to interact with other molecules, which may be related to the functional impairment of SOD3 upon A58T and R231G mutations, as well as their involvement in pathologies.

Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar ß-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed

Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae.

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar beta-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed.

Functional analysis of upstream activating elements in the promoter of the FBP1 gene from Saccharomyces cerevisiae.

We have investigated the effect of different carbon sources and of different mutations on the capacity of two elements, UAS1 and UAS2, from the promoter of the FBP1 gene to form specific DNA-protein complexes and to activate expression of a reporter gene. The complexes are observed with nuclear extracts from yeast derepressed on glycerol or ethanol. When hxk2 mutants are grown on glucose the nuclear extracts are able to complex UAS1 but not UAS2, while for wild-type cells grown on galactose only the complex with UAS2 is formed. In contrast, in vivo the operation of both UASs is high in ethanol, moderate to low in glycerol, and negligible in galactose; no expression is observed in glucose even in a hxk2 background. There is no effect of a MIG1 deletion, either in the formation of DNA-protein complexes or on the expression of reporter genes.

Modulation of trehalase activity in Saccharomyces cerevisiae by an intrinsic protein.

The regulation of cytosolic trehalase activity in yeast has been described as cycles of activation by phosphorylation by cAMP protein kinase. In this paper, evidence is presented for another regulatory mechanism--the binding of an endogenous inhibitory protein. This negative modulator was isolated during the purification procedure of cytosolic cryptic trehalase from repressed wild-type cells of Saccharomyces cerevisiae. However, in derepressed cells the inhibitor was not found nor was it present in ras2 mutant cells submitted to a heat treatment. The trehalase inhibitory activity proved to be a calmodulin ligand protein and, therefore, involved in the modulation of trehalase activity by Ca2+ ions.