Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Molecular details of INH-C-10 binding to wt KatG and Its S315T mutant

Isoniazid (INH) is still one of the two most effective antitubercular drugs and is included in all recommended multitherapeutic regimens. Because of the increasing resistance of Mycobacterium tuberculosis to INH, mainly associated with mutations in the katG gene, new INH-based compounds have been proposed to circumvent this problem. In this work, we present a detailed comparative study of the molecular determinants of the interactions between wt KatG or its S315T mutant form and either INH or INH-C10, a new acylated INH derivative. MD simulations were used to explore the conformational space of both proteins, and results indicate that the S315T mutation did not have a significant impact on the average size of the access tunnel in the vicinity of these residues. Our simulations also indicate that the steric hindrance role assigned to Asp137 is transient and that electrostatic changes can be important in understanding the enzyme activity data of mutations in KatG. Additionally, molecular docking studies were used to determine the preferred modes of binding of the two substrates. Upon mutation, the apparently less favored docking solution for reaction became the most abundant, suggesting that S315T mutation favors less optimal binding modes. Moreover, the aliphatic tail in INH-C10 seems to bring the hydrazine group closer to the heme, thus favoring the apparent most reactive binding mode, regardless of the enzyme form. The ITC data is in agreement with our interpretation of the C10 alkyl chain role and helped to rationalize the significantly lower experimental MIC value observed for INH-C10. This compound seems to be able to counterbalance most of the conformational restrictions introduced by the mutation, which are thought to be responsible for the decrease in INH activity in the mutated strain. Therefore, INH-C10 appears to be a very promising lead compound for drug development.