DDX3 DEAD-box RNA helicase plays a central role in mitochondrial protein quality control in Leishmania

Abstract

DDX3 is a highly conserved member of ATP-dependent DEAD-box RNA helicases with multiple functions in RNA metabolism and cellular signaling. Here, we describe a novel function for DDX3 in regulating the mitochondrial stress response in the parasitic protozoan Leishmania. We show that genetic inactivation of DDX3 leads to the accumulation of mitochondrial reactive oxygen species (ROS) associated with a defect in hydrogen peroxide detoxification. Upon stress, ROS production is greatly enhanced, causing mitochondrial membrane potential loss, mitochondrial fragmentation, and cell death. Importantly, this phenotype is exacerbated upon oxidative stress in parasites forced to use the mitochondrial oxidative respiratory machinery. Furthermore, we show that in the absence of DDX3, levels of major components of the unfolded protein response as well as of polyubiquitinated proteins increase in the parasite, particularly in the mitochondrion, as an indicator of mitochondrial protein damage. Consistent with these findings, immunoprecipitation and mass-spectrometry studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly the antioxidant response, the unfolded protein response, and the AAA-ATPase p97/VCP/Cdc48, which is essential in mitochondrial protein quality control by driving proteosomal degradation of polyubiquitinated proteins. Complementation studies using DDX3 deletion mutants lacking conserved motifs within the helicase core support that binding of DDX3 to ATP is essential for DDX3's function in mitochondrial proteostasis. As a result of the inability of DDX3-depleted Leishmania to recover from ROS damage and to survive various stresses in the host macrophage, parasite intracellular development was impaired. Collectively, these observations support a central role for the Leishmania DDX3 homolog in preventing ROS-mediated damage and in maintaining mitochondrial protein quality control.

Valosin-containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress

“…Polypeptides modified with K48‐linked polyubiquitin chains, which also serve as a major targeting signal for the proteasome (Chau et al, ), are physiological substrates for VCP. Our previous studies revealed Li VCP association with several proteasome subunits (Padmanabhan et al, ). The partial colocalisation of Li VCP with the ER and its weaker association with the mitochondrion, as suggested by indirect immunofluorescence studies, are in line with Li VCP being involved in the extraction of polyubiquitinated proteins from cellular organelles for subsequent proteasomal degradation, as described in other eukaryotes (Qi et al, ; Taylor & Rutter, ).…”

“…Furthermore, we tested whether parasite mutant lines with a significant decrease in VCP expression levels are more sensitive to proteasome inhibitors or to other stresses inducing proteotoxicity. Proteosome inhibition by MG‐132 is known to increase accumulation of polyubiquitinated proteins, also in Leishmania (Padmanabhan et al, ). Both the Li VCP (NEO/+) and the Li VCP (NEO/HYG) + VCP HA strains showed ~2‐fold higher sensitivity to MG‐132 than the WT strain (Figure S11a,c).…”

“…Proteosome inhibition by MG-132 is known to increase accumulation of polyubiquitinated proteins, also in Leishmania (Padmanabhan et al, 2016). Both the LiVCP (NEO/+) and the LiVCP (NEO/HYG) + VCP HA strains showed~2-fold higher sensitivity to MG-132 than the WT strain ( Figure S11a,c).…”

The DEAD-box RNA helicases and multiple abiotic stresses in plants: a systematic review of recent advances and challenges

“…Molecular processes such as replication, transcription, posttranscriptional modification, translation, mRNA splicing and mRNA export and its stability are vital for living organisms, and helicase enzymes are to play very crucial role in gene expression and regulation (Hartman et al, 2006;Bolinger et al, 2007;Bolinger et al, 2010;Górna et al, 2010;Ranji et al, 2011;Andreou and Klostermeier, 2013;Chu et al, 2016;Lin et al, 2016;Padmanabhan et al, 2016). Helicase enzymes are categorized into two types namely, DNA helicase and RNA helicase (Jankowsky, 2011).…”

The Pumilio-domain protein PUF6 contributes to SIDER2 retroposon-mediated mRNA decay in Leishmania

“…Clear supernatants were further incubated with Protein G anti-HA magnetic beads at 4°C for 4 h on a gentle rotator. Beads were then washed by TBS-0.05% Tween (SigmaAldrich) three times (30 sec each) by gentle agitation and subjected to LC-MS/MS analysis as previously described (Padmanabhan et al 2016). Immunoprecipitation experiments were done on cotransfected parasites as above except that prior to freezing the parasites in liquid nitrogen, they were exposed to 400 mJ/cm 2 UV irradiation on a Stratalinker 2400 UV crosslinker in PBS medium and then immediately harvested and snap-frozen.…”