TRNT-1 Deficiency Is Associated with Loss of tRNA Integrity and Imbalance of Distinct Proteins.

Mitochondrial diseases are a group of heterogeneous disorders caused by dysfunctional mitochondria. Interestingly, a large proportion of mitochondrial diseases are caused by defects in genes associated with tRNA metabolism. We recently discovered that partial loss-of-function mutations in tRNA Nucleotidyl Transferase 1 (TRNT1), the nuclear gene encoding the CCA-adding enzyme essential for modifying both nuclear and mitochondrial tRNAs, causes a multisystemic and clinically heterogenous disease termed SIFD (sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay; SIFD). However, it is not clear how mutations in a general and essential protein like TRNT1 cause disease with such clinically broad but unique symptomatology and tissue involvement. Using biochemical, cell, and mass spectrometry approaches, we demonstrate that TRNT1 deficiency is associated with sensitivity to oxidative stress, which is due to exacerbated, angiogenin-dependent cleavage of tRNAs. Furthermore, reduced levels of TRNT1 lead to phosphorylation of Eukaryotic Translation Initiation Factor 2 Subunit Alpha (eIF2α), increased reactive oxygen species (ROS) production, and changes in the abundance of distinct proteins. Our data suggest that the observed variable SIFD phenotypes are likely due to dysregulation of tRNA maturation and abundance, which in turn negatively affects the translation of distinct proteins.

Loss of PDCD4 contributes to enhanced chemoresistance in Glioblastoma multiforme through de-repression of Bcl-xL translation.

Glioblastoma multiforme (GBM) is the most common and aggressive form of tumor of the central nervous system. Despite significant efforts to improve treatments, patient survival rarely exceeds 18 months largely due to the highly chemoresistant nature of these tumors. Importantly, misregulation of the apoptotic machinery plays a key role in the development of drug resistance. We previously demonstrated that Bcl-xL, an important anti-apoptotic protein, is regulated at the level of translation by the tumor suppressor programmed cell death 4 (PDCD4). We report here a strong correlation between low expression of PDCD4 and high expression of Bcl-xL in adult de novo GBM, GBM tumor initiating cells, and established GBM cell lines. Importantly, high Bcl-xL expression correlated significantly with poor progression and patient survival. We demonstrate that re-expression of PDCD4 in GBM cells down-regulated Bcl-xL expression and decreased cell viability. Finally, we show that direct inhibition of Bcl-xL by small molecule antagonist ABT-737 sensitizes GBM cells to doxorubicin. Our results identify Bcl-xL as a novel marker of GBM chemoresistance and advocate for the combined use of Bcl-xL antagonists and existing chemotherapeutics as a treatment option for this aggressive tumor.

Tumor suppressor PDCD4 represses internal ribosome entry site-mediated translation of antiapoptotic proteins and is regulated by S6 kinase 2.

Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2), which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-x(L). We now show that the FGF-2-S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-x(L) messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-x(L) translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-x(L)) that mediate FGF-2-S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression.

Silencing of tachyzoite enolase 2 alters nuclear targeting of bradyzoite enolase 1 in Toxoplasma gondii.

In Toxoplasma gondii, an intracellular parasite of the phylum Apicomplexa, two isoforms of enolase (ENO1 and ENO2) are expressed in stage-specific manner. ENO2 is expressed only in rapidly growing tachyzoites, while ENO1 is in slowly growing bradyzoites. Interestingly, the localization of ENO1 and ENO2 in the nuclear compartment has suggested possible roles of the proteins in gene regulation and/or cell cycle. To understand the physiological role of ENO2 in T. gondii, the expression of ENO2 was silenced using a homologous gene silencing procedure. The introduction or expression of ENO2 dsRNA successfully silenced the expression of ENO2 at the levels of transcripts and proteins. While there was no change in the growth rate of both tachyzoites and bradyzoites, a subtle phenotypic change was observed in the localization of the ENO1 gene product in the bradyzoite stage.

Toxoplasma gondii: over-expression of lactate dehydrogenase enhances differentiation under alkaline conditions.

Toxoplasma gondii, an intracellular parasite, has two distinctive growth stages, namely rapidly growing tachyzoites and slowly growing bradyzoites. Here we report a unique physiological function of the last committed glycolytic enzyme of T. gondii, lactate dehydrogenase (TgLDH), which is present in two isoforms and expressed in a stage-specific manner. TgLDH1 is present in tachyzoites while TgLDH2 is found in bradyzoites. Using clonal transgenic parasites over-expressing either TgLDH1 or TgLDH2, we showed that the enzymatic activity, growth, and virulence of tachyzoites were unaffected by the presence of the recombinant protein. Interestingly, under alkaline conditions the presence of the recombinant TgLDH proteins increased the differentiation, as detected by the formation of cyst structures in vitro, while green fluorescent protein did not. The differentiation enhancement of the recombinant TgLDH1 and TgLDH2 strongly suggests that TgLDH1 and TgLDH2 have an important physiological function, in addition to being glycolytic enzymes and differentiation markers