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Acta Naturae 2023Mitochondrial ribosome assembly is a complex multi-step process involving many additional factors. Ribosome formation differs in various groups of organisms. However,...
Mitochondrial ribosome assembly is a complex multi-step process involving many additional factors. Ribosome formation differs in various groups of organisms. However, there are universal steps of assembly and conservative factors that have been retained in evolutionarily distant taxa. METTL17, the object of the current study, is one of these conservative factors involved in mitochondrial ribosome assembly. It is present in both bacteria and the mitochondria of eukaryotes, in particular mice and humans. In this study, we tested a hypothesis of putative METTL17 methyltransferase activity. MALDI-TOF mass spectrometry was used to evaluate the methylation of a putative METTL17 target - a 12S rRNA region interacting with METTL17 during mitochondrial ribosome assembly. The investigation of METTL17 and other mitochondrial ribosome assembly factors is of both fundamental and practical significance, because defects in mitochondrial ribosome assembly are often associated with human mitochondrial diseases.
PubMed: 38234605
DOI: 10.32607/actanaturae.25441 -
International Journal of Molecular... Dec 2023The combination of trastuzumab and pertuzumab as first-line therapy in patients with HER2-positive breast cancer has shown significant clinical benefits compared to...
The combination of trastuzumab and pertuzumab as first-line therapy in patients with HER2-positive breast cancer has shown significant clinical benefits compared to trastuzumab alone. However, despite initial therapeutic success, most patients eventually progress, and tumors develop acquired resistance and invariably relapse. Therefore, there is an urgent need to improve our understanding of the mechanisms governing resistance in order to develop targeted therapeutic strategies with improved efficacy. We generated four novel HER2-positive cell lines via prolonged exposure to trastuzumab and pertuzumab and determined their resistance rates. Long-term resistance was confirmed by a significant increase in the colony-forming capacity of the derived cells. We authenticated the molecular identity of the new lines via both immunohistochemistry for the clinical phenotype and molecular profiling of point mutations. HER2 overexpression was confirmed in all resistant cell lines, and acquisition of resistance to trastuzumab and pertuzumab did not translate into differences in ER, PR, and HER2 receptor expression. In contrast, changes in the expression and activity of other HER family members, particularly HER4, were observed. In the same vein, analyses of the receptor and effector kinase status of different cellular pathways revealed that the MAPK pathway may be involved in the acquisition of resistance to trastuzumab and pertuzumab. Finally, proteomic analysis confirmed a significant change in the abundance patterns of more than 600 proteins with implications in key biological processes, such as ribosome formation, mitochondrial activity, and metabolism, which could be relevant mechanisms in the generation of resistance in HER2-positive breast cancer. We concluded that these resistant BCCLs may be a valuable tool to better understand the mechanisms of acquisition of resistance to trastuzumab and pertuzumab-based anti-HER2 therapy.
Topics: Humans; Female; Trastuzumab; Breast Neoplasms; Proteomics; Neoplasm Recurrence, Local; Cell Line; Antibodies, Monoclonal, Humanized
PubMed: 38203378
DOI: 10.3390/ijms25010207 -
International Journal of Molecular... Dec 2023Mitochondria carry out various vital roles in eukaryotic cells, including ATP energy synthesis, the regulation of apoptosis, Fe-S cluster formation, and the metabolism...
Mitochondria carry out various vital roles in eukaryotic cells, including ATP energy synthesis, the regulation of apoptosis, Fe-S cluster formation, and the metabolism of fatty acids, amino acids, and nucleotides. Throughout evolution, mitochondria lost most of their ancestor's genome but kept the replication, transcription, and translation machinery. Protein biosynthesis in mitochondria is specialized in the production of highly hydrophobic proteins encoded by mitochondria. These proteins are components of oxidative phosphorylation chain complexes. The coordination of protein synthesis must be precise to ensure the correct assembly of nuclear-encoded subunits for these complexes. However, the regulatory mechanisms of mitochondrial translation in human cells are not yet fully understood. In this study, we examined the contribution of the SLIRP protein in regulating protein biosynthesis in mitochondria. Using a click-chemistry approach, we discovered that deletion of the gene disturbs mitochondrial translation, leading to the dysfunction of complexes I and IV, but it has no significant effect on complexes III and V. We have shown that this protein interacts only with the small subunit of the mitochondrial ribosome, which may indicate its involvement in the regulation of the mitochondrial translation initiation stage.
Topics: Humans; Electron Transport Complex IV; Mitochondrial Proteins; HEK293 Cells; Mitochondria; Eukaryotic Cells; RNA-Binding Proteins
PubMed: 38203264
DOI: 10.3390/ijms25010093 -
Molecular Cell Jan 2024Cellular proteostasis requires transport of polypeptides across membranes. Although defective transport processes trigger cytosolic rescue and quality control mechanisms...
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.
Topics: Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Protein Biosynthesis; Ribosomes; Peptide Hydrolases
PubMed: 38199007
DOI: 10.1016/j.molcel.2023.12.013 -
BioRxiv : the Preprint Server For... Dec 2023The efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A is primarily...
The efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A is primarily known as an elongation factor which binds ribosomes to alleviate ribosome stalling at sequences encoding polyprolines or combinations of proline with glycine and charged amino acids. eIF5A is known to impact the mitochondrial function across a variety of species although the precise molecular mechanism underlying this impact remains unclear. We found that depletion of eIF5A in yeast drives reduced translation and levels of TCA cycle and oxidative phosphorylation proteins. We further found that loss of eIF5A leads to the accumulation of mitoprotein precursors in the cytosol as well as to the induction of a mitochondrial import stress response. Here we identify an essential polyproline-containing protein as a direct eIF5A target for translation: the mitochondrial inner membrane protein Tim50, which is the receptor subunit of the TIM23 translocase complex. We show how eIF5A directly controls mitochondrial protein import through the alleviation of ribosome stalling along mRNA at the mitochondrial surface. Removal of the polyprolines from Tim50 rescues the mitochondrial import stress response, as well as the translation of oxidative phosphorylation reporter genes in an eIF5A loss of function. Overall, our findings elucidate how eIF5A impacts the mitochondrial function by reducing ribosome stalling and facilitating protein translation, thereby positively impacting the mitochondrial import process.
PubMed: 38187585
DOI: 10.1101/2023.12.19.572290 -
Scientific Reports Jan 2024Selective degradation of dysfunctional or excess mitochondria is a fundamental process crucial for cell homeostasis in almost all eukaryotes. This process relies on...
Selective degradation of dysfunctional or excess mitochondria is a fundamental process crucial for cell homeostasis in almost all eukaryotes. This process relies on autophagy, an intracellular self-eating system conserved from yeast to humans and is thus called mitophagy. Detailed mechanisms of mitophagy remain to be fully understood. Here we show that mitochondrial degradation in budding yeast, which requires the pro-mitophagic protein Atg32, is strongly reduced in cells lacking Egd1, a beta subunit of the nascent polypeptide-associated complex acting in cytosolic ribosome attachment and protein targeting to mitochondria. By contrast, loss of the sole alpha subunit Egd2 or the beta subunit paralogue Btt1 led to only a partial or slight reduction in mitophagy. We also found that phosphorylation of Atg32, a crucial step for priming mitophagy, is decreased in the absence of Egd1. Forced Atg32 hyperphosphorylation almost completely restored mitophagy in egd1-null cells. Together, we propose that Egd1 acts in Atg32 phosphorylation to facilitate mitophagy.
Topics: Autophagy-Related Proteins; Mitophagy; Peptides; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Mitochondria; Transcription Factors; DNA-Binding Proteins
PubMed: 38177147
DOI: 10.1038/s41598-023-50245-7 -
Scientific Reports Jan 2024Ovarian cancer (OVCA), a prevalent gynecological malignancy, ranks as the fourth most common cancer among women. Mitotic Arrest Deficient 2 Like 2 (MAD2L2), a...
Ovarian cancer (OVCA), a prevalent gynecological malignancy, ranks as the fourth most common cancer among women. Mitotic Arrest Deficient 2 Like 2 (MAD2L2), a chromatin-binding protein and a component of DNA polymerase ζ, has been previously identified as an inhibitor of tumor growth in colorectal cancer. However, the roles of MAD2L2 in OVCA, including its expression, impact, and prognostic significance, remain unclear. We employed bioinformatics tools, Cox Regression analysis, and in vitro cell experiments to investigate its biological functions. Our findings reveal that MAD2L2 typically undergoes genomic alterations, such as amplifications and deep deletions. Moreover, we observed an overexpression of MAD2L2 mRNA in OVCA patients, correlating with reduced survival rates, particularly in those with Grade IV tumors. Furthermore, analysis of mRNA biofunctions indicated that MAD2L2 is predominantly localized in the organellar ribosome, engaging mainly in NADH dehydrogenase activity. This was deduced from the results of gene ontology enrichment analysis, which also identified its role as a structural constituent in mitochondrial translation elongation. These findings were corroborated by KEGG pathway analysis, further revealing MAD2L2's involvement in tumor metabolism and the cell death process. Notably, MAD2L2 protein expression showed significant associations with various immune cells, including CD4+T cells, CD8+T cells, B cells, natural killer cells, and Myeloid dendritic cells. Additionally, elevated levels of MAD2L2 were found to enhance cell proliferation and migration in OVCA cells. The upregulation of MAD2L2 also appears to inhibit the ferroptosis process, coinciding with increased mTOR signaling activity in these cells. Our study identifies MAD2L2 as a novel regulator in ovarian tumor progression and offers new insights for treating OVCA.
Topics: Humans; Female; Ovarian Neoplasms; Proteins; Neoplastic Processes; Cell Proliferation; RNA, Messenger; Cell Line, Tumor; Mad2 Proteins
PubMed: 38167649
DOI: 10.1038/s41598-023-50744-7 -
RNA (New York, N.Y.) Feb 2024Mitochondria-associated RNA-binding proteins (RBPs) have emerged as key contributors to mitochondrial biogenesis and homeostasis. With few examples known, we set out to...
Mitochondria-associated RNA-binding proteins (RBPs) have emerged as key contributors to mitochondrial biogenesis and homeostasis. With few examples known, we set out to identify RBPs that regulate nuclear-encoded mitochondrial mRNAs (NEMmRNAs). Our systematic analysis of RNA targets of 150 RBPs identified RBPs with a preference for binding NEMmRNAs, including LARP4, a La RBP family member. We show that LARP4's targets are particularly enriched in mRNAs that encode respiratory chain complex proteins (RCCPs) and mitochondrial ribosome proteins (MRPs) across multiple human cell lines. Through quantitative proteomics, we demonstrate that depletion of LARP4 leads to a significant reduction in RCCP and MRP protein levels. Furthermore, we show that LARP4 depletion reduces mitochondrial function, and that LARP4 re-expression rescues this phenotype. Our findings shed light on a novel function for LARP4 as an RBP that binds to and positively regulates NEMmRNAs to promote mitochondrial respiratory function.
Topics: Humans; Cell Line; Mitochondria; RNA-Binding Proteins
PubMed: 38164626
DOI: 10.1261/rna.079799.123 -
International Journal of Molecular... Dec 2023Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they...
Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they either filter out scanning ribosomes or allow downstream translation initiation via leaky scanning or reinitiation. Previous reports concurred that eIF4G2, a long-known but insufficiently studied eIF4G1 homologue, can rescue the downstream translation, but disagreed on whether it is leaky scanning or reinitiation that eIF4G2 promotes. Here, we investigated a unique human mRNA that encodes two highly conserved proteins (POLGARF with unknown function and POLG, the catalytic subunit of the mitochondrial DNA polymerase) in overlapping reading frames downstream of a regulatory uORF. We show that the uORF renders the translation of both POLGARF and POLG mRNAs reliant on eIF4G2. Mechanistically, eIF4G2 enhances both leaky scanning and reinitiation, and it appears that ribosomes can acquire eIF4G2 during the early steps of reinitiation. This emphasizes the role of eIF4G2 as a multifunctional scanning guardian that replaces eIF4G1 to facilitate ribosome movement but not ribosome attachment to an mRNA.
Topics: Humans; RNA, Messenger; Peptide Chain Initiation, Translational; 5' Untranslated Regions; Ribosomes; Reading Frames; Open Reading Frames; Protein Biosynthesis; DNA Polymerase gamma
PubMed: 38138978
DOI: 10.3390/ijms242417149 -
Biomedicine & Pharmacotherapy =... Jan 2024Nullomers are the shortest strings of absent amino acid (aa) sequences in a species or group of species. Primes are those nullomers that have not been detected in the...
Nullomers are the shortest strings of absent amino acid (aa) sequences in a species or group of species. Primes are those nullomers that have not been detected in the genome of any species. 9S1R is a 5-aa peptide prime sequence attached to 5-arginine aa, used to treat triple negative breast cancer (TNBC) in an in vivo mouse model. This unique peptide, administered with a trehalose carrier (9S1R-NulloPT), offers enhanced solubility and exhibits distinct anti-cancer effects against TNBC. In our study, we investigated the effect of 9S1R-NulloPT on tumor growth, metabolism, metastatic burden, tumor immune-microenvironment (TME), and transcriptome of aggressive mouse TNBC tumors. Notably, treated mice had smaller tumors in the initial phase of the treatment, as compared to untreated control, and diminished in vivo and ex vivo bioluminescence at later-stages - indicative of metabolically quiescent, dying tumors. The treatment also caused changes in TME with increased infiltration of immune cells and altered tumor transcriptome, with 365 upregulated genes and 710 downregulated genes. Consistent with in vitro data, downregulated genes were enriched in cellular metabolic processes (179), specifically mitochondrial TCA cycle/oxidative phosphorylation (44), and translation machinery/ribosome biogenesis (45). The upregulated genes were associated with the developmental (13), ECM organization (12) and focal adhesion pathways (7). In conclusion, our study demonstrates that 9S1R-NulloPT effectively reduced tumor growth during its initial phase, altering the TME and tumor transcriptome. The treatment induced mitochondrial pathology which led to a metabolic deceleration in tumors, aligning with in vitro observations.
Topics: Humans; Mice; Animals; Triple Negative Breast Neoplasms; Cell Line, Tumor; Peptides; Mitochondria; Transcriptome; Tumor Microenvironment
PubMed: 38118350
DOI: 10.1016/j.biopha.2023.115997