Inherent metabolic preferences differentially regulate the sensitivity of Th1 and Th2 cells to ribosome-inhibiting antibiotics.

Mitochondrial translation is essential to maintain mitochondrial function and energy production. Mutations in genes associated with mitochondrial translation cause several developmental disorders, and immune dysfunction is observed in many such patients. Besides genetic mutations, several antibiotics targeting bacterial ribosomes are well-established to inhibit mitochondrial translation. However, the effect of such antibiotics on different immune cells is not fully understood. Here, we addressed the differential effect of mitochondrial translation inhibition on different subsets of helper T cells (Th) of mice and humans. Inhibition of mitochondrial translation reduced the levels of mitochondrially encoded electron transport chain subunits without affecting their nuclear-encoded counterparts. As a result, mitochondrial oxygen consumption reduced dramatically, but mitochondrial mass was unaffected. Most importantly, we show that inhibition of mitochondrial translation induced apoptosis, specifically in Th2 cells. This increase in apoptosis was associated with higher expression of Bim and Puma, two activators of the intrinsic pathway of apoptosis. We propose that this difference in the sensitivity of Th1 and Th2 cells to mitochondrial translation inhibition reflects the intrinsic metabolic demands of these subtypes. Though Th1 and Th2 cells exhibit similar levels of oxidative phosphorylation, Th1 cells exhibit higher levels of aerobic glycolysis than Th2 cells. Moreover, Th1 cells are more sensitive to the inhibition of glycolysis, while higher concentrations of glycolysis inhibitor 2-deoxyglucose are required to induce cell death in the Th2 lineage. These observations reveal that selection of metabolic pathways for substrate utilization during differentiation of Th1 and Th2 lineages is a fundamental process conserved across species.

Mitochondrial ribosome biogenesis and redox sensing.

Mitoribosome biogenesis is a complex process involving RNA elements encoded in the mitochondrial genome and mitoribosomal proteins typically encoded in the nuclear genome. This process is orchestrated by extra-ribosomal proteins, nucleus-encoded assembly factors, which play roles across all assembly stages to coordinate ribosomal RNA processing and maturation with the sequential association of ribosomal proteins. Both biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided insights into their assembly process. In this article, we will briefly outline the current understanding of mammalian mitoribosome biogenesis pathways and the factors involved. Special attention is devoted to the recent identification of iron-sulfur clusters as structural components of the mitoribosome and a small subunit assembly factor, the existence of redox-sensitive cysteines in mitoribosome proteins and assembly factors, and the role they may play as redox sensor units to regulate mitochondrial translation under stress.

Decoding the Enigma: Translation Termination in Human Mitochondria.

Mitochondrial translation is a complex process responsible for the synthesis of essential proteins involved in oxidative phosphorylation, a fundamental pathway for cellular energy production. Central to this process is the termination phase, where dedicated factors play a pivotal role in ensuring accurate and timely protein production. This review provides a comprehensive overview of the current understanding of translation termination in human mitochondria, emphasizing structural features and molecular functions of two mitochondrial termination factors mtRF1 and mtRF1a.

The Schizosaccharomyces pombe DEAD-box protein Mss116 is required for mitoribosome assembly and mitochondrial translation.

DEAD-box helicases are important players in mitochondrial gene expression, which is necessary for mitochondrial respiration. In this study, we characterized Schizosaccharomyces pombe Mss116 (spMss116), a member of the family of DEAD-box RNA helicases. Deletion of spmss116 in a mitochondrial intron-containing background significantly reduced the levels of mitochondrial DNA (mtDNA)-encoded cox1 and cob1 mRNAs and impaired mitochondrial translation, leading to a severe respiratory defect and a loss of cell viability during stationary phase. Deletion of mitochondrial introns restored the levels of cox1 and cob1 mRNAs to wide-type (WT) levels but could not restore mitochondrial translation and respiration in Δspmss116 cells. Furthermore, deletion of spmss116 in both mitochondrial intron-containing and intronless backgrounds impaired mitoribosome assembly and destabilization of mitoribosomal proteins. Our findings suggest that defective mitochondrial translation caused by deletion of spmss116 is most likely due to impaired mitoribosome assembly.

Role of Rmd9p in 3'-end processing of mitochondrial 15S rRNA in Saccharomyces cerevisiae.

Ribosome biogenesis, involving processing/assembly of rRNAs and r-proteins is a vital process. In Saccharomyces cerevisiae mitochondria, ribosomal small subunit comprises 15S rRNA (15S). While the 15S 5'-end processing uses Ccm1p and Pet127p, the mechanisms of the 3'-end processing remain unclear. We reveal involvement of Rmd9p in safeguarding/processing 15S 3'-end. Rmd9p deficiency results in a cleavage at a position 183 nucleotides upstream of 15S 3'-end, and in the loss of the 3'-minor domain. Rmd9p binds to the sequences in the 3'-end region of 15S, and a genetic interaction between rmd9 and dss1 indicates that Rmd9p regulates/limits mtEXO activity during the 3'-end spacer processing.

ZLN005 alleviates PBDE-47 induced impairment of mitochondrial translation and neurotoxicity through PGC-1α/ERRα axis.

Recent studies are identified the mitochondria as critical targets of 2, 2', 4, 4'-tetrabromodiphenyl ether (PBDE-47) induced neurotoxicity. This study aimed at examining the impact of PBDE-47 exposure on mitochondrial translation, and its subsequent effect on PBDE-47 neurotoxicity. The Sprague-Dawley (SD) rat model and neuroendocrine pheochromocytoma (PC12) cells were adopted for the measurements of mitochondrial ATP levels, mitochondrial translation products, and expressions of important mitochondrial regulators, such as required meiotic nuclear division 1 (RMND1), estrogen-related receptor α (ERRα), and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). To delve into the role of PGC-1α/ERRα axis in mitochondrial translation, 2-(4-tert-butylphenyl) benzimidazole (ZLN005) was employed. Both cellular and animal model results shown that PBDE-47 impeded PGC-1α/ERRα axis and mitochondrial translation. PBDE-47 suppressed mitochondrial function in rat hippocampus and PC12 cells by decreasing relative mitochondrial DNA (mtDNA) content, mitochondrial translation products, and mitochondrial ATP levels. Particularly, ZLN005 reversed PBDE-47 neurotoxicity by enhancing mitochondrial translation through activation of PGC-1α/ERRα axis, yet suppressing PGC-1α with siRNA attenuates its neuroprotective effect in vitro. In conclusion, this work highlights the importance of mitochondrial translation in PBDE-47 neurotoxicity by presenting results from cellular and animal models and suggests a potential therapeutic approach through activation of PGC-1α/ERRα axis. ENVIRONMENTAL IMPLICATION: PBDEs have attracted extensive attention because of their high lipophilicity, persistence, and detection levels in various environmental media. Increasing evidence has shown that neurodevelopmental disorders in children are associated with PBDE exposure. Several studies have also found that perinatal PBDE exposure can cause long-lasting neurobehavioral abnormalities in experimental animals. Our recent studies have also demonstrated the impact of PBDE-47 exposure on mitochondrial biogenesis and dynamics, leading to memory and neurobehavioral deficits. Therefore, we explore whether the pathological mechanism of PBDE-47-induced neurotoxicity involves the regulation of mitochondrial translation through the PGC-1α/ERRα axis.

Sls1 and Mtf2 mediate the assembly of the Mrh5C complex required for activation of cox1 mRNA translation.

Mitochondrial translation depends on mRNA-specific activators. In Schizosaccharomyces pombe, DEAD-box protein Mrh5, pentatricopeptide repeat (PPR) protein Ppr4, Mtf2, and Sls1 form a stable complex (designated Mrh5C) required for translation of mitochondrial DNA (mtDNA)-encoded cox1 mRNA, the largest subunit of the cytochrome c oxidase complex. However, how Mrh5C is formed and what role Mrh5C plays in cox1 mRNA translation have not been reported. To address these questions, we investigated the role of individual Mrh5C subunits in the assembly and function of Mrh5C. Our results revealed that Mtf2 and Sls1 form a subcomplex that serves as a scaffold to bring Mrh5 and Ppr4 together. Mrh5C binds to the small subunit of the mitoribosome (mtSSU), but each subunit could not bind to the mtSSU independently. Importantly, Mrh5C is required for the association of cox1 mRNA with the mtSSU. Finally, we investigated the importance of the signature DEAD-box in Mrh5. We found that the DEAD-box of Mrh5 is required for the association of Mrh5C and cox1 mRNA with the mtSSU. Unexpectedly, this motif is also required for the interaction of Mrh5 with other Mrh5C subunits. Altogether, our results suggest that Mrh5 and Ppr4 cooperate in activating the translation of cox1 mRNA. Our results also suggest that Mrh5C activates the translation of cox1 mRNA by promoting the recruitment of cox1 mRNA to the mtSSU.

Aging-induced tRNAGlu-derived fragment impairs glutamate biosynthesis by targeting mitochondrial translation-dependent cristae organization.

Mitochondrial cristae, infoldings of the mitochondrial inner membrane, undergo aberrant changes in their architecture with age. However, the underlying molecular mechanisms and their contribution to brain aging are largely elusive. Here, we observe an age-dependent accumulation of Glu-5'tsRNA-CTC, a transfer-RNA-derived small RNA (tsRNA), derived from nuclear-encoded tRNAGlu in the mitochondria of glutaminergic neurons. Mitochondrial Glu-5'tsRNA-CTC disrupts the binding of mt-tRNALeu and leucyl-tRNA synthetase2 (LaRs2), impairing mt-tRNALeu aminoacylation and mitochondria-encoded protein translation. Mitochondrial translation defects disrupt cristae organization, leading to damaged glutaminase (GLS)-dependent glutamate formation and reduced synaptosomal glutamate levels. Moreover, reduction of Glu-5'tsRNA-CTC protects aged brains from age-related defects in mitochondrial cristae organization, glutamate metabolism, synaptic structures, and memory. Thus, beyond illustrating a physiological role for normal mitochondrial cristae ultrastructure in maintaining glutamate levels, our study defines a pathological role for tsRNAs in brain aging and age-related memory decline.

A kinetic dichotomy between mitochondrial and nuclear gene expression processes.

Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS subunit biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared with nuclear mRNAs, mt-mRNAs were produced 1,100-fold more, degraded 7-fold faster, and accumulated to 160-fold higher levels. Quantitative modeling and depletion of mitochondrial factors LRPPRC and FASTKD5 identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a 100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation.

Identification of TMEM126A as OXA1L-interacting protein reveals cotranslational quality control in mitochondria.

Cellular proteostasis requires transport of polypeptides across membranes. Although defective transport processes trigger cytosolic rescue and quality control mechanisms that clear translocases and membranes from unproductive cargo, proteins that are synthesized within mitochondria are not accessible to these mechanisms. Mitochondrial-encoded proteins are inserted cotranslationally into the inner membrane by the conserved insertase OXA1L. Here, we identify TMEM126A as a OXA1L-interacting protein. TMEM126A associates with mitochondrial ribosomes and translation products. Loss of TMEM126A leads to the destabilization of mitochondrial translation products, triggering an inner membrane quality control process, in which newly synthesized proteins are degraded by the mitochondrial iAAA protease. Our data reveal that TMEM126A cooperates with OXA1L in protein insertion into the membrane. Upon loss of TMEM126A, the cargo-blocked OXA1L insertase complexes undergo proteolytic clearance by the iAAA protease machinery together with its cargo.

Ribosome-targeting antibiotic control NLRP3-mediated inflammation by inhibiting mitochondrial DNA synthesis.

While antibiotics are designed to target bacteria specifically, most are known to affect host cell physiology. Certain classes of antibiotics have been reported to have immunosuppressive effects, but the underlying mechanisms remain elusive. Here, we show that doxycycline, a ribosomal-targeting antibiotic, effectively inhibited both mitochondrial translation and nucleotide-binding domain and leucine-rich repeat-containing protein 3 (NLRP3) inflammasome-mediated caspase-1 activation and interleukin-1ß (IL-1ß) production in bone-marrow-derived macrophages (BMDMs). In addition, knockdown of mitochondrial methionyl-tRNA formyltransferase (Mtfmt), which is rate limiting for mitochondrial translation, also resulted in the inhibition of NLRP3 inflammasome-mediated caspase-1 activation and IL-1ß secretion. Furthermore, both doxycycline treatment and Mtfmt knockdown blocked the synthesis of mitochondrial DNA (mtDNA) and the generation of oxidized mtDNA (Ox-mtDNA), which serves as a ligand for NLRP3 inflammasome activation. In addition, in vivo results indicated that doxycycline mitigated NLRP3 inflammasome-dependent inflammation, including lipopolysaccharide-induced systemic inflammation and endometritis. Taken together, the results unveil the antibiotics targeting the mitoribosome have the ability to mitigate NLRP3 inflammasome activation by inhibiting mitochondrial translation and mtDNA synthesis thus opening up new possibilities for the treatment of NLRP3-related diseases.

GTPBP8 is required for mitoribosomal biogenesis and mitochondrial translation.

Mitochondrial translation occurs on the mitochondrial ribosome, also known as the mitoribosome. The assembly of mitoribosomes is a highly coordinated process. During mitoribosome biogenesis, various assembly factors transiently associate with the nascent ribosome, facilitating the accurate and efficient construction of the mitoribosome. However, the specific factors involved in the assembly process, the precise mechanisms, and the cellular compartments involved in this vital process are not yet fully understood. In this study, we discovered a crucial role for GTP-binding protein 8 (GTPBP8) in the assembly of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. GTPBP8 is identified as a novel GTPase located in the matrix and peripherally bound to the inner mitochondrial membrane. Importantly, GTPBP8 is specifically associated with the mt-LSU during its assembly. Depletion of GTPBP8 leads to an abnormal accumulation of mt-LSU, indicating that GTPBP8 is critical for proper mt-LSU assembly. Furthermore, the absence of GTPBP8 results in reduced levels of fully assembled 55S monosomes. This impaired assembly leads to compromised mitochondrial translation and, consequently, impaired mitochondrial function. The identification of GTPBP8 as an important player in these processes provides new insights into the molecular mechanisms underlying mitochondrial protein synthesis and its regulation.

D-Allulose Ameliorates Dysregulated Macrophage Function and Mitochondrial NADH Homeostasis, Mitigating Obesity-Induced Insulin Resistance.

D-allulose, a rare sugar, has been proposed to have potential benefits in addressing metabolic disorders such as obesity and type 2 diabetes (T2D). However, the precise mechanisms underlying these effects remain poorly understood. We aimed to elucidate the mechanisms by which D-allulose influences obesity-induced insulin resistance. We conducted gene set enrichment analysis on the liver and white adipose tissue of mice exposed to a high-fat diet (HFD) along with the white adipose tissue of individuals with obesity. Our study revealed that D-allulose effectively suppressed IFN-γ, restored chemokine signaling, and enhanced macrophage function in the livers of HFD-fed mice. This implies that D-allulose curtails liver inflammation, alleviating insulin resistance and subsequently impacting adipose tissue. Furthermore, D-allulose supplementation improved mitochondrial NADH homeostasis and translation in both the liver and white adipose tissue of HFD-fed mice. Notably, we observed decreased NADH homeostasis and mitochondrial translation in the omental tissue of insulin-resistant obese subjects compared to their insulin-sensitive counterparts. Taken together, these results suggest that supplementation with allulose improves obesity-induced insulin resistance by mitigating the disruptions in macrophage and mitochondrial function. Furthermore, our data reinforce the crucial role that mitochondrial energy expenditure plays in the development of insulin resistance triggered by obesity.

Molecular Investigation of Mitochondrial RNA19 Role in the Pathogenesis of MELAS Disease.

In mammalian mitochondria, the processing of primary RNA transcripts involves a coordinated series of cleavage and modification events, leading to the formation of processing intermediates and mature mt-RNAs. RNA19 is an unusually stable unprocessed precursor, physiologically polyadenylated, which includes the 16S mt-rRNA, the mt-tRNALeuUUR and the mt-ND1 mRNA. These peculiarities, together with the alteration of its steady-state levels in cellular models with defects in mitochondrial function, make RNA19 a potentially important molecule for the physiological regulation of mitochondrial molecular processes as well as for the pathogenesis of mitochondrial diseases. In this work, we quantitatively and qualitatively examined RNA19 in MELAS trans-mitochondrial cybrids carrying the mtDNA 3243A>G transition and displaying a profound mitochondrial translation defect. Through a combination of isokinetic sucrose gradient and RT-qPCR experiments, we found that RNA19 accumulated and co-sedimented with the mitoribosomal large subunit (mt-LSU) in mutant cells. Intriguingly, exogenous expression of the isolated LARS2 C-terminal domain (Cterm), which was shown to rescue defective translation in MELAS cybrids, decreased the levels of mt-LSU-associated RNA19 by relegating it to the pool of free unbound RNAs. Overall, the data reported here support a regulatory role for RNA19 in mitochondrial physiopathological processes, designating this RNA precursor as a possible molecular target in view of therapeutic strategy development.

Chronic inflammation in high-fat diet-fed mice: Unveiling the early pathogenic connection between liver and adipose tissue.

Obesity-induced chronic inflammation has been linked to several autoimmune diseases, including rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. The underlying mechanisms are not yet fully understood, but it is believed that chronic inflammation in adipose tissue can lead to the production of pro-inflammatory cytokines and chemokines, which can trigger immune responses and contribute to the development of autoimmune diseases. However, the underlying mechanisms that lead to the infiltration of immune cells into adipose tissue are not fully understood. In this study, we observed a time-dependent response to a high-fat diet in the liver and epididymal white adipose tissue using gene set enrichment analysis. Our findings revealed a correlation between early abnormal innate immune responses in the liver and late inflammatory response in the adipose tissue, that eventually leads to systemic inflammation. Specifically, our data suggest that the dysregulated NADH homeostasis in the mitochondrial matrix, interacting with the mitochondrial translation process, could serve as a sign marking the transition from liver inflammation to adipose tissue inflammation. Taken together, our study provides valuable insights into the molecular mechanisms underlying the development of chronic inflammation and associated autoimmune diseases in obesity.

RNA binding protein: coordinated expression between the nuclear and mitochondrial genomes in tumors.

Mitochondria are the only organelles regulated by two genomes. The coordinated translation of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA), which together co-encode the subunits of the oxidative phosphorylation (OXPHOS) complex, is critical for determining the metabolic plasticity of tumor cells. RNA-binding protein (RBP) is a post-transcriptional regulatory factor that plays a pivotal role in determining the fate of mRNA. RBP rapidly and effectively reshapes the mitochondrial proteome in response to intracellular and extracellular stressors, mediating the cytoplasmic and mitochondrial translation balance to adjust mitochondrial respiratory capacity and provide energy for tumor cells to adapt to different environmental pressures and growth needs. This review highlights the ability of RBPs to use liquid-liquid phase separation (LLPS) as a platform for translation regulation, integrating nuclear-mitochondrial positive and retrograde signals to coordinate cross-department translation, reshape mitochondrial energy metabolism, and promote the development and survival of tumor cells.

Insulin-Degrading Enzyme Interacts with Mitochondrial Ribosomes and Respiratory Chain Proteins.

Insulin-degrading enzyme (IDE) is a highly conserved metalloprotease that is mainly localized in the cytosol. Although IDE can degrade insulin and some other low molecular weight substrates efficiently, its ubiquitous expression suggests additional functions supported by experimental findings, such as a role in stress responses and cellular protein homeostasis. The translation of a long full-length IDE transcript has been reported to result in targeting to mitochondria, but the role of IDE in this compartment is unknown. To obtain initial leads on the function of IDE in mitochondria, we used a proximity biotinylation approach to identify proteins interacting with wild-type and protease-dead IDE targeted to the mitochondrial matrix. We find that IDE interacts with multiple mitochondrial ribosomal proteins as well as with proteins involved in the synthesis and assembly of mitochondrial complex I and IV. The mitochondrial interactomes of wild type and mutant IDE are highly similar and do not reveal any likely proteolytic IDE substrates. We speculate that IDE could adopt similar additional non-proteolytic functions in mitochondria as in the cytosol, acting as a chaperone and contributing to protein homeostasis and stress responses.

Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway.

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.

Discovery of Mitochondrial Ribosomes.

In this introductory chapter, I will briefly describe how I came to discover the mammalian mitoribosome and will add a few notes on my contribution to the field.

Mitoribosome Biogenesis.

Mitoribosome biogenesis is a complex and energetically costly process that involves RNA elements encoded in the mitochondrial genome and mitoribosomal proteins most frequently encoded in the nuclear genome. The process is catalyzed by extra-ribosomal proteins, nucleus-encoded assembly factors that act in all stages of the assembly process to coordinate the processing and maturation of ribosomal RNAs with the hierarchical association of ribosomal proteins. Biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided hints regarding their assembly. In this general concept chapter, we will briefly describe the current knowledge, mainly regarding the mammalian mitoribosome biogenesis pathway and factors involved, and will emphasize the biological sources and approaches that have been applied to advance the field.