Mitochondrial methionyl-tRNA Formyltransferase Research Articles

Tumor-derived mitochondrial formyl peptides suppress tumor immunity through modification of the tumor microenvironment.

Mitochondrial N-formylpeptides are released from damaged or dead cells to the extracellular spaces and cause inflammatory responses. The role of mitochondrial N-formylpeptides in aseptic systemic inflammatory response syndromes induced by trauma or cardiac surgery has been well investigated. However, there are no reports regarding the role of mitochondrial N-formylpeptides in cancer. In this study, we investigated the role of tumor cell-derived mitochondrial N-formylpeptides in anti-tumor immunity using knockout murine tumor cells of mitochondrial methionyl-tRNA formyltransferase (MTFMT), which catalyze N-formylation of mitochondrial DNA-encoded proteins. There was no apparent difference among the wild-type and MTFMT-knockout clones of E.G7-OVA cells with respect to morphology, mitochondrial dynamics, glycolysis and oxidative phosphorylation, oxygen consumption rate, or in vitro cell growth. In contrast, in vivo tumor growth of MTFMT-knockout cells was slower than that of wild-type cells. A reduced number of myeloid-derived suppressor cells and an increase of cytotoxic T-lymphocytes in the tumor tissues were observed in the MTFMT-knockout tumors. These results suggested that tumor cell-derived mitochondrial N-formylpeptides had a negative role in the host anti-tumor immunity through modification of the tumor microenvironment.

Methionine Improves Boar Sperm Quality by Promoting Mitochondrial Translation during Liquid Storage.

Boar sperm quality serves as an important indicator of reproductive efficiency, playing a direct role in enhancing the output of livestock production. It has been demonstrated that mitochondrial protein translation is present in sperm and plays a crucial role in regulating sperm motility, capacitation and in vitro fertilization rate. The present study aimed to determine whether methionine supplementation enhances mitochondrial translation in boar sperm, thereby improving sperm quality. The results showed a significant elevation in the abundance of mitochondrial methionyl-tRNA formyltransferase (MTFMT), a crucial enzyme for mitochondrial protein translation, and mitochondrial DNA-encoded cytochrome c oxidase subunit 1 (COX1) in boar sperm exhibiting high motility. Both amino acids and methionine supplementation significantly enhanced boar sperm motility during storage. Moreover, methionine supplementation mitigates the loss of acrosomal integrity, enhances the expression of COX1, and boosts mitochondrial activity. Furthermore, the positive impact of methionine was negated in the presence of the mitochondrial translation inhibitor chloramphenicol. Together, these findings suggest that boar sperm may utilize methionine as a protein translation substrate to enhance sperm motility by stimulating mitochondrial protein translation. The supplementation of methionine may enhance the quality of boar sperm, thereby providing guidance for the optimization of diluent formulations for liquid storage and the identification of physiological regulators that regulate sperm motility.

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-1β (IL-1β) 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-1β 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.

Mitochondrial Methionyl-tRNA Formyltransferase Deficiency Alleviates Metaflammation by Modulating Mitochondrial Activity in Mice

Various studies have revealed the association of metabolic diseases with inflammation. Mitochondria are key organelles involved in metabolic regulation and important drivers of inflammation. However, it is uncertain whether the inhibition of mitochondrial protein translation results in the development of metabolic diseases, such that the metabolic benefits related to the inhibition of mitochondrial activity remain unclear. Mitochondrial methionyl-tRNA formyltransferase (Mtfmt) functions in the early stages of mitochondrial translation. In this study, we reveal that feeding with a high-fat diet led to the upregulation of Mtfmt in the livers of mice and that a negative correlation existed between hepatic Mtfmt gene expression and fasting blood glucose levels. A knockout mouse model of Mtfmt was generated to explore its possible role in metabolic diseases and its underlying molecular mechanisms. Homozygous knockout mice experienced embryonic lethality, but heterozygous knockout mice showed a global reduction in Mtfmt expression and activity. Moreover, heterozygous mice showed increased glucose tolerance and reduced inflammation, which effects were induced by the high-fat diet. The cellular assays showed that Mtfmt deficiency reduced mitochondrial activity and the production of mitochondrial reactive oxygen species and blunted nuclear factor-κB activation, which, in turn, downregulated inflammation in macrophages. The results of this study indicate that targeting Mtfmt-mediated mitochondrial protein translation to regulate inflammation might provide a potential therapeutic strategy for metabolic diseases.

Leigh syndrome followed by parkinsonism in an adult with homozygous c.626C>T mutation in MTFMT.

ObjectiveTo report the clinical, radiologic, biochemical, and molecular characteristics in a 46-year-old participant with adult-onset Leigh syndrome (LS), followed by parkinsonism.MethodsCase description with diagnostic workup included blood and CSF analysis, skeletal muscle investigations, blue native polyacrylamide gel electrophoresis, whole exome sequencing targeting nuclear genes involved in mitochondrial transcription and translation, cerebral MRI, 123I-FP-CIT brain single-photon emission computed tomography (SPECT), and C-11 raclopride positron emission tomography (PET).ResultsThe participant was found to have a defect in the oxidative phosphorylation caused by a c.626C>T mutation in the gene coding for mitochondrial methionyl-tRNA formyltransferase (MTFMT), which is a pathogenic mutation affecting intramitochondrial protein translation. The proband had a normal concentration of lactate in blood and no abnormal microscopic findings in skeletal muscle. Cerebral MRI showed bilateral lesions in the striatum, mesencephalon, pons, and medial thalamus. Lactate concentration in CSF was increased. FP-CIT SPECT and C-11 raclopride PET demonstrated a defect in the dopaminergic system.ConclusionsWe report on a case with adult-onset LS related to a MTFMT mutation. Two years after the onset of symptoms of LS, the proband developed a parkinson-like disease. The c.626C>T mutation is the most common pathogenic mutation found in 22 patients reported earlier in the literature with a defect in MTFMT. The age of the previously reported cases varied between 14 months and 24 years. Our report expands the phenotypical spectrum of MTFMT-related neurologic disease and provides clinical evidence for involvement of MTFMT in extrapyramidal syndromes.

Mitochondrial methionyl N-formylation affects steady-state levels of oxidative phosphorylation complexes and their organization into supercomplexes

N-Formylation of the Met-tRNAMet by the nuclearly encoded mitochondrial methionyl-tRNA formyltransferase (MTFMT) has been found to be a key determinant of protein synthesis initiation in mitochondria. In humans, mutations in the MTFMT gene result in Leigh syndrome, a progressive and severe neurometabolic disorder. However, the absolute requirement of formylation of Met-tRNAMet for protein synthesis in mammalian mitochondria is still debated. Here, we generated a Mtfmt-KO mouse fibroblast cell line and demonstrated that N-formylation of the first methionine via fMet-tRNAMet by MTFMT is not an absolute requirement for initiation of protein synthesis. However, it differentially affected the efficiency of synthesis of mtDNA-coded polypeptides. Lack of methionine N-formylation did not compromise the stability of these individual subunits but had a marked effect on the assembly and stability of the OXPHOS complexes I and IV and on their supercomplexes. In summary, N-formylation is not essential for mitochondrial protein synthesis but is critical for efficient synthesis of several mitochondrially encoded peptides and for OXPHOS complex stability and assembly into supercomplexes.

Parkinson's disease: SNCA-, PARK2-, and LRRK2- targeting microRNAs elevated in cingulate gyrus

IntroductionIn order to better understand the role of epigenetic influences in the etiology of Parkinson's disease (PD), we studied the expression of microRNAs in gyri cinguli of patients and controls. MethodsExpression profiling of 744 well-characterized microRNAs in gyri cinguli from patients and controls using TaqMan array microRNA cards. Verification of significantly dysregulated microRNAs by SYBR Green qRT-PCR. ResultsFirst screen by TaqMan array identified 43 microRNAs that were upregulated in gyri cinguli from patients. Of those microRNAs, 13 are predicted to regulate at least one of six genes mutated in monogenic forms of PD (DJ-1, PARK2, PINK1, LRRK2, SNCA, and HTRA2). Five of these 13 microRNAs (-144, -199b, -221, -488, -544) were also found upregulated by SYBR Green qRT-PCR and are predicted to regulate either SNCA, PARK2, LRRK2 or combinations thereof. Consistently, expression of SNCA, PARK2, and LRRK2 was reduced in patients. An additional 5 out of ten potential target genes tested were downregulated. These are DRAM (DNA damage regulated autophagy modulator 1), predicted to be regulated by miR-144, EVC (Ellis Van Creveld Protein) by miR-221, ZNF440 (Zinc Finger Protein 440) by miR-199b, MTFMT (Mitochondrial Methionyl-tRNA Formyltransferase) by miR-488 and XIRP2 (Xin Actin Binding Repeat Containing) possibly controlled by miR-544a. ConclusionThe study identified five microRNAs that play a role in the etiology of Parkinson's disease likely by modifying expression of SNCA, PARK2, LRRK2 and additional genes required for normal cellular function.

Biochemical Characterization of Pathogenic Mutations in Human Mitochondrial Methionyl-tRNA Formyltransferase

N-Formylation of initiator methionyl-tRNA (Met-tRNA(Met)) by methionyl-tRNA formyltransferase (MTF) is important for translation initiation in bacteria, mitochondria, and chloroplasts. Unlike all other translation systems, the metazoan mitochondrial system is unique in using a single methionine tRNA (tRNA(Met)) for both initiation and elongation. A portion of Met-tRNA(Met) is formylated for initiation, whereas the remainder is used for elongation. Recently, we showed that compound heterozygous mutations within the nuclear gene encoding human mitochondrial MTF (mt-MTF) significantly reduced mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and Leigh syndrome in two unrelated patients. Patient P1 has a stop codon mutation in one of the MTF genes and an S209L mutation in the other MTF gene. P2 has a S125L mutation in one of the MTF genes and the same S209L mutation as P1 in the other MTF gene. Here, we have investigated the effect of mutations at Ser-125 and Ser-209 on activities of human mt-MTF and of the corresponding mutations, Ala-89 or Ala-172, respectively, on activities of Escherichia coli MTF. The S125L mutant has 653-fold lower activity, whereas the S209L mutant has 36-fold lower activity. Thus, both patients depend upon residual activity of the S209L mutant to support low levels of mitochondrial protein synthesis. We discuss the implications of these and other results for whether the effect of the S209L mutation on mitochondrial translational efficiency is due to reduced activity of the mutant mt-MTF and/or reduced levels of the mutant mt-MTF.

A genome-wide association study reveals a novel candidate gene for sperm motility in pigs

Sperm motility is one of the most widely used parameters in order to evaluate boar semen quality. However, this trait can only be measured after puberty. Thus, the use of genomic information appears as an appealing alternative to evaluate and improve selection for boar fertility traits earlier in life. With this study we aimed to identify SNPs with significant association with sperm motility in two different commercial pig populations and to identify possible candidate genes within the identified QTL regions. We performed a single-SNP genome-wide association study using genotyped animals from a Landrace-based (L1) and a Large White-based (L2) pig populations. For L1, a total of 602 animals genotyped for 42,551 SNPs were used in the association analysis. For L2, a total of 525 animals genotyped for 40,890 SNPs were available. After the association analysis, a false discovery rate q-value ≤0.05 was used as the threshold for significant association. No SNPs were significantly associated with sperm motility in L1, while six SNPs on Sus scrofa chromosome 1 (position 117.26–119.56Mb) were significant in L2. The mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene, which affects translation efficiency of proteins in sperm cells, was identified as a putative candidate gene. The significant markers identified in this study may be useful to enhance the genetic improvement of sperm motility by selection of boars at an earlier age under a marker assisted selection strategy.

Clinical and functional characterisation of the combined respiratory chain defect in two sisters due to autosomal recessive mutations in MTFMT

Exome sequencing identified compound heterozygous mutations in the recently discovered mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene in two sisters with mild Leigh syndrome and combined respiratory chain deficiency. The mutations lead to undetectable levels of the MTFMT protein. Blue native polyacrylamide gel electrophoresis showed decreased complexes I and IV, and additional products stained with complex V antibodies, however the overall steady state level of mt-tRNAMet was normal. Our data illustrate that exome sequencing is an excellent diagnostic tool, and its value in clinical medicine is enormous, however it can only be optimally exploited if combined with detailed phenotyping and functional studies.

Mutations in MTFMT Underlie a Human Disorder of Formylation Causing Impaired Mitochondrial Translation

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of Met-tRNA(Met) formylation.

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNA(f)(Met)). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNA(f)(Met), as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNA(f)(Met) to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNA(f)(Met). This accessory factor might be unnecessary when formylated Met-tRNA(f)(Met) is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Delta fmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNA(f)(Met) due to loss of the MIS1 gene that encodes the mitochondrial C(1)-tetrahydrofolate synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.

S.11.02 The ubiquitin/proteasome system in neurodegenerative disease

In order to better understand the role of epigenetic influences in the etiology of Parkinson's disease (PD), we studied the expression of microRNAs in gyri cinguli of patients and controls.Expression profiling of 744 well-characterized microRNAs in gyri cinguli from patients and controls using TaqMan array microRNA cards. Verification of significantly dysregulated microRNAs by SYBR Green qRT-PCR.First screen by TaqMan array identified 43 microRNAs that were upregulated in gyri cinguli from patients. Of those microRNAs, 13 are predicted to regulate at least one of six genes mutated in monogenic forms of PD (DJ-1, PARK2, PINK1, LRRK2, SNCA, and HTRA2). Five of these 13 microRNAs (-144, -199b, -221, -488, -544) were also found upregulated by SYBR Green qRT-PCR and are predicted to regulate either SNCA, PARK2, LRRK2 or combinations thereof. Consistently, expression of SNCA, PARK2, and LRRK2 was reduced in patients. An additional 5 out of ten potential target genes tested were downregulated. These are DRAM (DNA damage regulated autophagy modulator 1), predicted to be regulated by miR-144, EVC (Ellis Van Creveld Protein) by miR-221, ZNF440 (Zinc Finger Protein 440) by miR-199b, MTFMT (Mitochondrial Methionyl-tRNA Formyltransferase) by miR-488 and XIRP2 (Xin Actin Binding Repeat Containing) possibly controlled by miR-544a.The study identified five microRNAs that play a role in the etiology of Parkinson's disease likely by modifying expression of SNCA, PARK2, LRRK2 and additional genes required for normal cellular function.

Mammalian Mitochondrial Initiation Factor 2 Supports Yeast Mitochondrial Translation without Formylated Initiator tRNA

Initiation of protein synthesis in mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl-tRNA (fMet-tRNAfMet) in a process involving initiation factor 2 (IF2). However, yeast strains disrupted at the FMT1 locus, encoding mitochondrial methionyl-tRNA formyltransferase, lack detectable fMet-tRNAfMet but exhibit normal mitochondrial function as evidenced by normal growth on non-fermentable carbon sources. Here we show that mitochondrial translation products in Saccharomyces cerevisiae were synthesized in the absence of formylated initiator tRNA. ifm1 mutants, lacking the mitochondrial initiation factor 2 (mIF2), are unable to respire, indicative of defective mitochondrial protein synthesis, but their respiratory defect could be complemented by plasmid-borne copies of either the yeast IFM1 gene or a cDNA encoding bovine mIF2. Moreover, the bovine mIF2 sustained normal respiration in ifm1 fmt1 double mutants. Bovine mIF2 supported the same pattern of mitochondrial translation products as yeast mIF2, and the pattern did not change in cells lacking formylated Met-tRNAfMet. Mutant yeast lacking any mIF2 retained the ability to synthesize low levels of a subset of mitochondrially encoded proteins. The ifm1 null mutant was used to analyze the domain structure of yeast mIF2. Contrary to a previous report, the C terminus of yeast mIF2 is required for its function in vivo, whereas the N-terminal domain could be deleted. Our results indicate that formylation of initiator methionyl-tRNA is not required for mitochondrial protein synthesis. The ability of bovine mIF2 to support mitochondrial translation in the yeast fmt1 mutant suggests that this phenomenon may extend to mammalian mitochondria as well.