Effects of Antiretroviral Drugs on the Glutathione and Glucose Metabolism of Cultured Brain Cells

The highly active antiretroviral therapy (HAART) has been successfully used for 30 years to treat the aquired immuno deficiency syndrome (AIDS) and the human immuno deficiency virus (HIV)-associated dementia. However, as minor neurocognitive deficits persist in treated HIV patients potential adverse consequences of antiretroviral drugs on brain cells are currently intensively discussed, but lack sufficient experimental proof. To address such questions, this thesis investigated the acute effects of various antiretroviral drugs from the classes of reverse transcriptase (RT) inhibitors and protease inhibitors on viability, glutathione (GSH) metabolism and glycolytic flux of brain cells, using primary cultures of astrocytes and neurons as model systems. A treatment with protease and RT inhibitors did not acutely damage cultured brain cells. However, the incubation of viable cultured astrocytes or neurons with protease inhibitors, but not with RT inhibitors, strongly stimulated cellular GSH release. The protease inhibitor-induced acceleration of GSH export was completely blocked by an inhibitor of the multidrug resistance protein 1 (Mrp1), suggesting that this exporter mediates the protease inhibitor-induced GSH depletion of brain cells. Protease inhibitors or RT inhibitors did not modulate the glycolytic flux of cultured astrocytes. In contrast, 8-hydroxy efavirenz (8-OH-efv), the primary metabolite of the frequently used RT inhibitor efavirenz, accelerated the glycolysis-derived lactate release from viable cultured astrocytes. However, in contrast to respiratory chain inhibitors, a direct inhibition of mitochondrial respiration by 8-OH-efv appears not to be the mechanism underlying the 8-OH-efv-mediated stimulation of glycolytic flux in astrocytes. As the lifelong treatment of HIV patients with antiretroviral drugs establishes a chronic exposure of brain cells to such compounds or their metabolites, alterations in basic metabolism of brain cells, such as those reported in this thesis, should be considered to contribute to the reported mild neurocognitive impairments of treated HIV patients

Effekte von Antiretroviralen Therapeutika auf den Glutahion- und Glucosestoffwechsel von Gehirnzellkulturen

The highly active antiretroviral therapy (HAART) has been successfully used for 30 years to treat the aquired immuno deficiency syndrome (AIDS) and the human immuno deficiency virus (HIV)-associated dementia. However, as minor neurocognitive deficits persist in treated HIV patients potential adverse consequences of antiretroviral drugs on brain cells are currently intensively discussed, but lack sufficient experimental proof. To address such questions, this thesis investigated the acute effects of various antiretroviral drugs from the classes of reverse transcriptase (RT) inhibitors and protease inhibitors on viability, glutathione (GSH) metabolism and glycolytic flux of brain cells, using primary cultures of astrocytes and neurons as model systems. A treatment with protease and RT inhibitors did not acutely damage cultured brain cells. However, the incubation of viable cultured astrocytes or neurons with protease inhibitors, but not with RT inhibitors, strongly stimulated cellular GSH release. The protease inhibitor-induced acceleration of GSH export was completely blocked by an inhibitor of the multidrug resistance protein 1 (Mrp1), suggesting that this exporter mediates the protease inhibitor-induced GSH depletion of brain cells. Protease inhibitors or RT inhibitors did not modulate the glycolytic flux of cultured astrocytes. In contrast, 8-hydroxy efavirenz (8-OH-efv), the primary metabolite of the frequently used RT inhibitor efavirenz, accelerated the glycolysis-derived lactate release from viable cultured astrocytes. However, in contrast to respiratory chain inhibitors, a direct inhibition of mitochondrial respiration by 8-OH-efv appears not to be the mechanism underlying the 8-OH-efv-mediated stimulation of glycolytic flux in astrocytes. As the lifelong treatment of HIV patients with antiretroviral drugs establishes a chronic exposure of brain cells to such compounds or their metabolites, alterations in basic metabolism of brain cells, such as those reported in this thesis, should be considered to contribute to the reported mild neurocognitive impairments of treated HIV patients

Crystal structure of the C-terminal 2',5'-phosphodiesterase domain of group A rotavirus protein VP3

In response to viral infections, the mammalian innate immune system induces the production of the second messenger 2'-5' oligoadenylate (2-5A) to activate latent ribonuclease L (RNase L) that restricts viral replication and promotes apoptosis. A subset of rotaviruses and coronaviruses encode 2',5'-phosphodiesterase enzymes that hydrolyze 2-5A, thereby inhibiting RNase L activation. We report the crystal structure of the 2',5'-phosphodiesterase domain of group A rotavirus protein VP3 at 1.39 Å resolution. The structure exhibits a 2H phosphoesterase fold and reveals conserved active site residues, providing insights into the mechanism of 2-5A degradation in viral evasion of host innate immunity

Molecular architecture of LSM14 interactions involved in the assembly of mRNA silencing complexes

The LSM domain-containing protein LSM14/Rap55 plays a role in mRNA decapping, translational repression, and RNA granule (P-body) assembly. How LSM14 interacts with the mRNA silencing machinery, including the eIF4E-binding protein 4E-T and the DEAD-box helicase DDX6, is poorly understood. Here we report the crystal structure of the LSM domain of LSM14 bound to a highly conserved C-terminal fragment of 4E-T. The 4E-T C-terminus forms a bi-partite motif that wraps around the N-terminal LSM domain of LSM14. We also determined the crystal structure of LSM14 bound to the C-terminal RecA-like domain of DDX6. LSM14 binds DDX6 via a unique non-contiguous motif with distinct directionality as compared to other DDX6-interacting proteins. Together with mutational and proteomic studies, the LSM14-DDX6 structure reveals that LSM14 has adopted a divergent mode of binding DDX6 in order to support the formation of mRNA silencing complexes and P-body assembly

Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs

Meiosis arrest female 1 (MARF1) is a cytoplasmic RNA binding protein that is essential for meiotic progression of mouse oocytes, in part by limiting retrotransposon expression. MARF1 is also expressed in somatic cells and tissues; however, its mechanism of action has yet to be investigated. Human MARF1 contains a NYN-like domain, two RRMs and eight LOTUS domains. Here we provide evidence that MARF1 post-transcriptionally silences targeted mRNAs. MARF1 physically interacts with the DCP1:DCP2 mRNA decapping complex but not with deadenylation machineries. Importantly, we provide a 1.7 Å resolution crystal structure of the human MARF1 NYN domain, which we demonstrate is a bona fide endoribonuclease, the activity of which is essential for the repression of MARF1-targeted mRNAs. Thus, MARF1 post-transcriptionally represses gene expression by serving as both an endoribonuclease and as a platform that recruits the DCP1:DCP2 decapping complex to targeted mRNAs

Molecular mechanism of the RNA helicase DHX37 and its activation by UTP14A in ribosome biogenesis

Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD). Structural comparisons of the nucleotide-free DHX37-RNA complex with DEAH helicases bound to RNA and ATP analogs reveal conformational changes resulting in a register shift in the bound RNA, suggesting a mechanism for ATP-dependent 3′–5′ RNA translocation. We further show that a conserved sequence motif in UTP14A interacts with and activates DHX37 by stimulating its ATPase activity and enhancing RNA binding. In turn, the CTD of DHX37 is required, but not sufficient, for interaction with UTP14A in vitro and is essential for ribosome biogenesis in vivo. Together, these results shed light on the mechanism of DHX37 and the function of UTP14A in controlling its recruitment and activity during ribosome biogenesis.ISSN:1355-8382ISSN:1469-900