Mitochondrial Ribosomal Protein Research Articles

A New Oomycete Metabarcoding Method Using the rps10 Gene

Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause plant and animal diseases. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have evaluated the mitochondrial 40S ribosomal protein S10 ( rps10) gene as a locus for oomycete metabarcoding and provide primers predicted to amplify this region from all oomycetes based on a wide range of available reference sequences. We evaluated its utility relative to the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Observed sequences and predicted taxonomy of ASVs and OTUs were compared with the known composition of the mock community. Both rps10 and ITS yielded ASVs with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1-bp difference. Taxonomic classifications of ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest that the rps10 locus results in substantially less amplification of nontarget organisms than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing.

Study on ubiquitination of proteins of the MRPS18 family in vitro

Aim. It is known that in cancerous cells of childhood tumors the pathological changes often include inactivation of the TP53 and RB-E2F1 cellular pathways. One of the proteins controlling the latter pathway is MRPS18-2, that belongs to a family of mitochondrial ribosomal proteins MRPS18. It is important, to study the stability of proteins of this family and their ubiquitination, that might help to conclude about the functional properties of these proteins and their role in cell transformation. Methods. Cloning of cDNA in FLAG vector for expression of fusion proteins, transfection of human tumor cells MCF7, study on cellular localization of MRPS18 family proteins and their ubiquitination by fluorescence microscopy, using specific antibodies. Results. The FLAG-MRPS18-1 and FLAG-MRPS18-3 fusion proteins are partially co-localizing with the HA-Ub fusion protein in the cytoplasm of MCF7 cells. The FLAG-MRPS18-2 protein is localized also in the nucleus. Conclusions. Nuclear localization of the FLAG-MRPS18-2 protein may indicate its additional functions in the cell: due to the interaction with the RB protein and the positive effect on mono-ubiquitination of histone H2B, the MRPS18-2 protein may be involved in the regulation of chromatin structure.

Abstract P1036: Role Of Mitochondrial Ribosomal Protein L7/l12 (mrpl12) In Diabetic Ischemic Heart Disease

Myocardial infarction (MI) is a significant cause of death in diabetic patients. In addition, growing evidence suggests that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood. The current study aimed to investigate the role of mitochondrial ribosomal protein L7/L12 (MRPL12) in mouse models of type II diabetes (db/db mice)and high-fat diet (HFD) mice with or without induction of MI and human hearts with or without diabetes (n=7) .Data analysis revealed an increase in MRPL12 levels in the LV tissue of HFD fed mice with MI than in LV tissues of low-fat diet-fed mice with MI, whereas MRPL12 levels remained unchanged in the db/db mice with MI. Intriguingly, we found increased MRPL12 levels in atrial appendage tissue of diabetic patients with ischemic heart disease compared to non-diabetic patients. We utilized human cardiomyocyte cell-line (AC-16) as surrogate models to delineate the mechanisms; surprisingly, adenovirus-mediated overexpression of MRPL12 with or without hyperglycemia in AC-16 cardiomyocytes does not affect mitochondrial OXPHOS . In addition, overexpression of MRPL12 had no effect on the mitochondrial ROS, mitochondrial membrane depolarization, and caspase activity in AC-16 cardiomyocytes. Whereas RNA interference (RNAi)-mediated MRPL12 silencing remarkedly reduced mitochondrial oxidative phosphorylation in AC-16 cells without any stress. In addition, knockdown of MRPL12 increased mitochondrial membrane depolarization mitochondrial ROS and reduced maximal respiratory capacity of mitochondria without any stress. Overall, our results provide new insights into the role of MRPL12 in the pathophysiology of MI in diabetes.

Quantitative Proteomic Assessment of Key Proteins Regulated by Electrical Pulse-mediated Galloflavin Delivery in Triple-Negative Breast Cancer Cells

Triple-negative breast cancer (TNBC) is the most lethal subset of breast cancers, lacking targeted therapies. There is a critical need to identify alternative treatments for TNBC. Towards this, in this research, we investigated the anticancer effects of Galloflavin (GF), an LDHB inhibitor, in reducing the proliferation of MDA-MB-231, the human triple-negative breast cancer cells. To enhance the uptake of GF, we applied electrical pulses (EP) with GF and studied its protein profile characteristics and viability. We used GF at a concentration of 100μM with 800V/cm, 100μs, and eight electrical pulses to treat these cells. Label-free, high throughput, quantitative proteomics results indicated that 172 proteins were significantly downregulated, while 222 proteins were significantly upregulated. The upregulated proteins include Cytochrome C Oxidase Assembly Factor and Mitochondrial Ribosomal proteins. Key downregulated proteins include LDHB, and ENO1 in EP+GF treatments, compared to GF only, indicating the effect of EP+GF combination in reducing the proliferation of the TNBC cells. These results pave the path for additional therapy for TNBC and the various pathways the TNBC cells proceeded with Electrochemotherapy.

Selected Articles from This Issue

Ewing sarcoma is characterized by oncogenic gene fusions and elevated lysine-specific demethylase-1 (LSD1) expression. While Theisen and colleagues previously devised a small molecule LSD1 inhibitor that demonstrates therapeutic efficacy against Ewing sarcoma — SP-2509 — treatment with SP-2509 alone potentiates drug resistance. In this study, Tokarsky et al., sought to elucidate molecular mechanisms underlying SP-2509 resistance by using a CRISPR–Cas9 loss-of-function screen in SP-2509–sensitive Ewing sarcoma cell lines. Results from the screen demonstrated that abrogating expression of mitochondrial ribosomal protein L45 (MRPL45) as well as members of electron transport chain complexes III (CIII) and IV (CIV) reduces SP-2509 responsiveness. Accordingly, CIII inhibitors and targeted removal of genes encoding CIII constituents ubiquinone-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (UQCRFS1) and cytochrome c1 (CYC1) using CRISPR–Cas9 induces SP-2509 resistance in Ewing sarcoma cell lines. Silencing UQCRFS1, CYC1, or MRPL45 expression diminishes transcriptional responses to SP-2509, indicating that mitochondrial function contributes to transcriptional programs underlying SP-2509 cytotoxicity. Overall, this study implicates mitochondrial dysfunction as a mechanism facilitating SP-2509 resistance, providing a target that may augment SP-2509 efficacy.Germline BRCA1/2 mutations increase the risk of multiple cancers that exhibit homologous recombination deficiency (HRD). BRCA1/2 mutations and subsequent HRD are associated with a number of genomic mutational signatures that have been previously described and seem to represent use of backup DNA repair pathways. These signatures, such as HRDetect and others, have been quantified and used to inform prognoses and therapeutic strategies. In this study, Moore and colleagues used current pre-clinical knowledge about polymerase theta-mediated end joining (TMEJ) and the genomic scars most typical of this backup repair pathway to determine if TMEJ signatures have prognostic value in advanced ovarian cancer. The authors found that both a TMEJ deletion signature with 4 or more base pair stretches of microhomology (TMEJ4) and TMEJ-associated templated insertions (TINS) are enriched in tumors with BRCA1/2 mutations. The authors also discovered that patients displaying high HRDetect and TINS signatures survive significantly longer than patients displaying one or neither signature. Taken together, this study highlights the potential utility of TMEJ mutational signatures in advanced ovarian cancer prognosis.Immune checkpoint inhibitors (ICI) are ineffective in pancreatic cancer, rendering combination treatments capable of augmenting ICI imperative. Radiation and inhibition of DNA damage response proteins, such as DNA-dependent protein kinase (DNA-PK), can activate antitumor immunity. However, whether and how DNA-PK inhibition mediates antitumor immunity in typically immunology “cold” cancers, including pancreatic cancer, is unknown. In their study, Wang and colleagues used radiation and DNA-PK inhibitor M3814 in mouse pancreatic cancer models to test whether the combination regimen can enhance antitumor immunity. The authors found that radiation-M3814 treatment induces type I interferon (T1IFN) signaling and slows tumor growth uniquely in mice with intact immune systems. The authors demonstrated that radiation and M3814-mediated T1IFN signaling is dependent on RNA polymerase III (POL III)-retinoic acid-inducible gene I (RIG-I)-mitochondrial anti-viral-signaling protein (MAVS) signaling, presumably through production and recognition of double-stranded RNA. Furthermore, T1IFN signaling induces tumor PD-L1 expression, and radiation-M3814 treatment augments anti-PD-L1 efficacy in vivo. In sum, this study elucidates a novel mechanism by which ICI efficacy may be enhanced in typically immunologically “cold” pancreatic cancers.Increased MYC stabilization is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), but molecular mechanisms underlying MYC stabilization in T-ALL are incompletely understood. To study effects of two MYC mutations — T58A and S62A — on MYC stabilization and MYC-induced pathophysiology in T-ALL, Daniel and colleagues generated T-cell–specific MycWT, MycT58A, and MycS62A knock-in mouse models in which endogenous wild-type Myc can be maintained or eliminated. The authors observed that MycT58A induces heightened ex vivo T-cell proliferation and thymic mass relative to MycS62A, and that MycT58A causes T-cell lymphomagenesis more rapidly than does MycS62A. Nonetheless, MycS62A does induce clonal T-cell lymphomas that resemble T-ALL and, unlike MycT58A, causes non-transgene driven, incidental thymic B-cell lymphomas with splenomegaly. Differential phenotypes engendered by MycT58A and MycS62A are accompanied by disparate transcriptional programs. MycT58A is associated with altered expression of genes underlying angiogenesis and inflammation, and MycS62A displays changes in expression of genes involved in maintaining genomic stability. Altogether, this study demonstrates that MycT58A and MycS62A induce unique pathophysiology in T-cell lymphomas, which may inform future research and T-ALL disease management strategies.

Expression and prognosis analysis of mitochondrial ribosomal protein family in breast cancer

Breast cancer (BC) is characterized by high morbidity. Mitochondrial ribosomal protein (MRP) family participates in mitochondrial energy metabolism, underlying BC progression. This study aims to analyze the expression and prognosis effect of the MRP genes in BC patients. GEPIA2, UALCAN, cBioPortal, and MethSurv were used to demonstrate the differential expression, genomic alteration profiles, and DNA methylation of the MRP gene family in BC. Functional enrichment analysis and protein–protein interaction network construction were performed to understand the biological function. Based on 1056 TCGA samples with the transcriptional level of MRPs, Kaplan–Meier curves, Cox, and LASSO regression were applied to explore their prognostic effects. 12 MRPs were upregulated in BC, which were associated with gene amplification and DNA methylation. MRP genetic alteration occurred in 42% of BC patients, and amplification was the most frequent variation. Functioning in its entirety, the MRP family was involved in mitochondrial translational termination, elongation, translation, and poly(A) RNA binding. High expression of MRPL1, MRPL13, MRPS6, MRPS18C, and MRPS35, as well as low levels of MRPL16, and MRPL40 significantly indicated poor prognosis in BC patients. Thus, a novel MRP-based prognostic nomogram was established and verified with favorable discrimination and calibration. We not only provided a thorough expression and prognosis analysis of the MRP family in BC patients but also constructed an MRP-based prognostic nomogram. It was suggested that MRPs acted as biomarkers in individualized risk prediction and may serve as potential therapeutic targets in BC patients.

Development of transcriptomics-based growth rate indices in two model eukaryotes and relevance to metatranscriptomics datasets.

Growth rate estimation is important to understand the flow of energy and nutrient elements in an ecosystem, but it has remained challenging, especially on microscopic organisms. In this study, we propose four growth rate indices that use mRNA abundance ratios between nuclear and mitochondrial genes: (1) total nuclear and mitochondrial mRNA ratio (Nuc:Mito‐TmRNA); (2) nuclear and mitochondrial ribosomal protein mRNA ratio (Nuc:Mito‐RPmRNA); (3) gene ontology (GO) terms and total mitochondrial mRNA ratios; and (4) nuclear and mitochondrial specific gene mRNA ratio. We examine these proposed ratios using RNA‐Seq datasets of Daphnia magna, and Saccharomyces cerevisiae retrieved from the NCBI Short Read Archive. The results showed that both Nuc:Mito‐TmRNA and Nuc:Mito‐RPmRNA ratio indices showed significant correlations with the growth rate for both species. A large number of GO terms mRNA ratios showed significant correlations with the growth rate of S. cerevisiae. Lastly, we identified mRNA ratios of several specific nuclear and mitochondrial gene pairs that showed significant correlations. We foresee future implications for the proposed mRNA ratios used in metatranscriptome analyses to estimate the growth rate of communities and species.

18β-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells.

BACKGROUNDGastric carcinoma (GC) is a common gastrointestinal malignancy worldwide. Based on the cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets. AIMTo explore the mechanism by which 18β-glycyrrhetinic acid (18β-GRA) regulates mitochondrial ribosomal protein L35 (MRPL35) related signal proteins to inhibit the proliferation of GC cells. METHODSCell counting kit-8 assay was used to detect the effects of 18β-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823. The apoptosis and cell cycle were assessed by flow cytometry. Cell invasion and migration were evaluated by Transwell assay, and cell scratch test was used to detect cell migration. Furthermore, a tumor model was established by hypodermic injection of 2.5 × 106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18β-GRA on GC cell proliferation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect MRPL35 expression in the engrafted tumors in mice. We used the term tandem mass tag (TMT) labeling combined with liquid chromatography–tandem mass spectrometry to screen for differentially expressed proteins (DEPs) extracted from GC cells and control cells after 18β-GRA intervention. A detailed bioinformatics analysis of these DEPs was performed, including Gene Ontology annotation and enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and so on. Moreover, STRING database (https://string-db.org/) was used to predict protein-protein interaction (PPI) relationships and Western blot was used to detect the expression of proteins of interest in GC cells. RESULTSThe results indicated that 18β-GRA could inhibit the proliferation of GC cells in a dose- and time-dependent manner. It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase. The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose, and the migration and invasiveness of GC cells were inhibited. The results of animal experiments showed that 18β-GRA could inhibit tumor formation in BALB/c nude mice, and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice. Using TMT technology, 609 DEPs, among which 335 were up-regulated and 274 were down-regulated, were identified in 18β-GRA intervention compared with control. We found that the intervention of 18β-GRA in GC cells involved many important biological processes and signaling pathways, such as cellular processes, biological regulation, and TP53 signaling pathway. Notably, after the drug intervention, MRPL35 expression was significantly down-regulated (P = 0.000247), TP53 expression was up-regulated (P = 0.02676), and BCL2L1 was down-regulated (P = 0.01699). Combined with the Retrieval of Interacting Genes/Proteins database, we analyzed the relationship between MRPL35, TP53, and BCL2L1 signaling proteins, and we found that COPS5, BAX, and BAD proteins can form a PPI network with MRPL35, TP53, and BCL2L1. Western blot analysis confirmed the intervention effect of 18β-GRA on GC cells, MRPL35, TP53, and BCL2L1 showed dose-dependent up/down-regulation, and the expression of COPS5, BAX, and BAD also increased/decreased with the change of 18β-GRA concentration.CONCLUSION18β-GRA can inhibit the proliferation of GC cells by regulating MRPL35, COPS5, TP53, BCL2L1, BAX, and BAD.

P53 regulates mitochondrial biogenesis via transcriptionally induction of mitochondrial ribosomal protein L12

The well-documented tumor suppressor p53 is also a major stress response factor for its diverse regulation on cellular energetics. However, the effect of p53 on mitochondrial biogenesis, which plays a predominant role in response to the elevated energy demands, appears to be pleiotropic in various conditions and has not reached agreement. Mitochondrial ribosomal protein L12 (MRPL12), reported as a bi-functional protein for its roles in both mitochondrial ribosomes and transcriptional complexes, is a core regulatory component in mitochondrial biogenesis. Here we proved that MRPL12 is transcriptionally regulated by p53. Furthermore, the p53/MRPL12 regulation of mitochondria is part of the signaling pathway that maintains the basal mitochondrial content and positively coordinates the mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) in response to metabolic perturbation. Since p53 serves as the‘Guardian of the Genome’, our findings may revealed a new mechanism underlying the conditions when more ATP is warranted to maintain the genome integrity and cell survival. Therefore the pharmacological intervention or metabolic modulation (e.g., through fasting or exercise) of the p53/MRPL12 pathway promises to be a therapeutic approach that can safeguard health.

Antagonistic regulatory effects of a single cis-acting expression quantitative trait locus between transcription and translation of the MRPL43 gene

BackgroundHeterogeneity of expression quantitative trait locus (eQTL) effects have been shown across gene expression processes. Knowledge on how to produce the heterogeneity is quite limited. This study aims to examine fluctuations in differential gene expression by alleles of sequence variants across expression processes.ResultsGenome-wide eQTL analyses with transcriptome-wide gene expression data revealed 20 cis-acting eQTLs associated simultaneously with mRNA expression, ribosome occupancy, and protein abundance. A 97 kb-long eQTL signal for mitochondrial ribosomal protein L43 (MRPL43) covered the gene, showing a heterogeneous effect size on gene products across expression stages. One allele of the eQTL was associated with increased mRNA expression and ribosome occupancy but decreased protein abundance. We examined the heterogeneity and found that the eQTL can be attributed to the independent functions of three nucleotide variants, with a strong linkage. NC_000010.11:g.100987606G > T, upstream of MRPL43, may regulate the binding affinity of transcription factors. NC_000010.11:g.100986746C > G, 3 bp from an MRPL43 splice donor site, may alter the splice site. NC_000010.11:g.100978794A > G, in the isoform with a long 3′-UTR, may strengthen the binding affinity of the microRNA. Individuals with the TGG haplotype at these three variants had higher levels of mRNA expression and ribosome occupancy than individuals with the GCA haplotype but lower protein levels, producing the flipped effect throughout the expression process.ConclusionsThese findings suggest that multiple functional variants in a linkage exert their regulatory functions at different points in the gene expression process, producing a complexity of single eQTLs.

Daf-16 mediated repression of cytosolic ribosomal protein genes facilitates a hypoxia sensitive to hypoxia resistant transformation in long-lived germline mutants.

In C. elegans, germline ablation leads to long life span and stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We demonstrate here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity in germline mutants occur independently of germline signals and instead rely on signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or “super-resistance”, to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that genes involved in both cytosolic and mitochondrial protein translation are repressed in sterile adults and further repressed only in germline deficient mutants as they age. Importantly, mutation of daf-16 specifically blocked the repression of cytosolic ribosomal protein genes, but not mitochondrial ribosomal protein genes, implicating DAF-16/FoxO mediated repression of cytosolic ribosomal protein genes as a mechanism of hypoxia super-resistance. Consistent with this hypothesis, the hypoxia super-resistance of aging germline deficient adults was also suppressed by dual mutation of ncl-1 and larp-1, two regulators of protein translation and ribosomal protein abundance. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are derived from developmentally dependent DAF-16/FoxO mediated repression of genes involved in cytosolic protein translation.

Characterization of Post‐translational Modifications of Mitochondrial RNA Polymerase and Mitochondrial Ribosomal Protein L12

Mitochondria play an important role in energy production and cellular metabolism. Mitochondria contain their own DNA (mtDNA), which encodes 13 subunits necessary for oxidative phosphorylation. Over 1500 other mitochondrial proteins, including the 77 remaining subunits required for oxidative phosphorylation and the machinery required for transcription and translation of mtDNA, are encoded by the nuclear genome. Thus, the nuclear and mitochondrial genomes must communicate to respond to the energetic needs of the cell. The mechanism of this communication is unclear. The mitochondrial proteome, including the transcriptional machinery, is subject to post‐translational modifications (PTMs) such as phosphorylation of serine, threonine, and tyrosine and acylation of lysine. We hypothesize that PTMs of the mitochondrial transcriptional machinery regulate mitochondrial gene expression, akin to mechanisms controlling nuclear gene expression. Transcription of mtDNA requires three nuclear‐encoded proteins: mitochondrial transcription factor A (TFAM), transcription factor B2 (TFB2M), and mitochondrial RNA polymerase (POLRMT). An accessory factor, mitochondrial ribosomal protein L12 (MRPL12), is thought to stabilize POLRMT and may promote transcription. Prior experiments in our lab show phosphorylation mimics of TFB2M have significantly reduced binding affinity for mtDNA and exhibit transcription initiation defects in vitro. Using mass spectrometry POLRMT was previously shown to be acetylated at one lysine and phosphorylated at nine amino acids, while MRPL12 contains five acetylated lysines and one phosphorylated threonine. The biochemical function of these modifications is unknown. PTMs were studied by using site‐directed mutagenesis to replace the amino acid of interest to mimic acetylation (lysine to glutamine) or phosphorylation (threonine to glutamate) of POLRMT and MRPL12. Mutated proteins were purified and their mtDNA promoter binding affinity was determined by fluorescence anisotropy experiments. Fluorescence anisotropy revealed that POLRMT PTM mimics had little effect on mtDNA binding. However, when WT MRPL12 was co‐incubated with WT POLRMT mtDNA binding affinity was enhanced by 30%. Increased binding affinity was lost with MRPL12 PTM mimics. WT and PTM mimics of MRPL12 were also overexpressed in mammalian cell lines. mtDNA transcript levels and mtDNA content were measured using quantitative PCR. mtDNA content was largely unchanged while some transcript‐dependent effects were observed in the presence of MRPL12 protein mutants.

Mitoribosomal defects aggravate liver cancer via aberrant glycolytic flux and T cell exhaustion

BackgroundMitochondria are involved in cancer energy metabolism, although the mechanisms underlying the involvement of mitoribosomal dysfunction in hepatocellular carcinoma (HCC) remain poorly understood. Here, we investigated the effects of mitoribosomal...

Bioenergetic and Metabolic Adaptation in Tumor Progression and Metastasis.

The ability of cancer cells to adjust their metabolism in response to environmental changes is a well-recognized hallmark of cancer. Diverse cancer and non-cancer cells within tumors compete for metabolic resources. Metabolic demands change frequently during tumor initiation, progression and metastasis, challenging our quest to better understand tumor biology and develop novel therapeutics. Vascularization, physical constraints, immune responses and genetic instability promote tumor evolution resulting in immune evasion, opportunities to breach basement membrane barriers and spread through the circulation and lymphatics. In addition, the unfolded protein response linked to the ubiquitin proteasome system is a key player in addressing stoichiometric imbalances between nuclear and mitochondrially-encoded protein subunits of respiratory complexes, and nuclear-encoded mitochondrial ribosomal protein subunits. While progressive genetic changes, some of which affect metabolic adaptability, contribute to tumorigenesis and metastasis through clonal expansion, epigenetic changes are also important and more dynamic in nature. Understanding the role of stromal and immune cells in the tumor microenvironment in remodeling cancer cell energy metabolism has become an increasingly important area of research. In this perspective, we discuss the adaptations made by cancer cells to balance mitochondrial and glycolytic energy metabolism. We discuss how hypoxia and nutrient limitations affect reductive and oxidative stress through changes in mitochondrial electron transport activity. We propose that integrated responses to cellular stress in cancer cells are central to metabolic flexibility in general and bioenergetic adaptability in particular and are paramount in tumor progression and metastasis.

Mitochondrial ribosomal protein genes connected with Alzheimer’s and tellurite toxicity

Mitochondrial diseases are a group of genetic disorders characterized by dysfunctional mitochondria. Within eukaryotic cells, mitochondria contain their own ribosomes, which synthesize small amounts of proteins, all of which are essential for the biogenesis of the oxidative phosphorylation system. The ribosome is an evolutionarily conserved macromolecular machine in nature both from a structural and functional point of view, universally responsible for the synthesis of proteins. Among the diseases afflicting humans, those of ribosomal origin – either cytoplasmic ribosomes (80S) or mitochondrial ribosomes (70S) – are relevant. These are inherited or acquired diseases most commonly caused by either ribosomal protein haploinsufficiency or defects in ribosome biogenesis. Here we review the scientific literature about the recent advances on changes in mitochondrial ribosomal structural and assembly proteins that are implicated in primary mitochondrial diseases and neurodegenerative disorders, and their possible connection with metalloid pollution and toxicity, with a focus on MRPL44, NAM9 (MNA6) and GEP3 (MTG3), whose lack or defect was associated with resistance to tellurite. Finally, we illustrate the suitability of yeast Saccharomyces cerevisiae (S. cerevisiae) and the nematode Caenorhabditis elegans (C. elegans) as model organisms for studying mitochondrial ribosome dysfunctions including those involved in human diseases.

Complimentary transcriptomic-metallomic analysis identifies risk of relapse for clear-cell renal cell carcinoma (ccRCC) patients.

378 Background: Patients with localized or locally advanced ccRCC undergo surgical resection by partial or radical nephrectomy. Approximately 50% of tumors recur within five years after surgery. Currently the only FDA approved drug in the adjuvant setting is sunitinib. However, the absence of an overall survival benefit and the prevalence of side effects limit widespread use of sunitinib. Accurate identification of patients at high risk of recurrence would enhance targeted adjuvant therapy, while sparing low-risk patients from side effects. A single scoring system, transcriptomic signature or genomic classifier, is unlikely to accurately define prognosis. Here, we describe an integrated transcriptomic-metallomic classifier to assess the risk of relapse in localized ccRCC. Methods: We investigated the transcriptomic landscape of Caucasian male patients with stage III ccRCCs who remained disease free (S3DF) or relapsed (S3RL) within 24 months after surgery using the TCGA Firehose Legacy cohort. In a separate cohort of S3RL and S3DF ccRCCs, copper (Cu) content and molecular distribution were analyzed by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS). Results: Transcriptomic analysis identified that S3RL tumors were enriched for genes encoding subunits of mitochondrial electron transport chain (ETC) and mitochondrial ribosomal proteins (MRPs), an indication that mitochondrial activity may contribute to the relapse. In contrast, S3DF tumors were enrichedfor genes related to immune responses, including genes encoding members ofMHC-II, suggesting tumor control by the immune system. We established a setof 23 genes (23G) as a signature that stratified ccRCCsinto distinct groups characterized by low, intermediateand high risk of relapse after surgery. Importantly, SEC-ICP-MS analysis identified augmented distribution of Cu to the Cu cytochrome c oxidase complex (Cu-COX) in tumor mitochondria from S3RL, consistent with the prediction of increased ETC activity in the transcriptomic analysis. Conclusions: Integration of the 23G transcriptomic signature and Cu-COX complex measurements in locoregional ccRCC can serve as a prognostic biomarker for recurrence, and as a predictive classifier for novel treatments, including inhibitors of ETC for the S3RL tumors. In particular, a combination classifier using both measurements may have a stronger prognostic/predictive power as compared to either biomarker alone.

TMT-Based Quantitative Proteomics Analysis Reveals the Panoramic Pharmacological Molecular Mechanism of β-Elemonic Acid Inhibition of Colorectal Cancer

Colorectal cancer (CRC) is one of the most common cancers worldwide but has limited available therapeutic methods; therefore, there is a need to develop highly efficient prevention and treatment strategies. Here, we investigated the anti-cancer activity of β-elemonic acid (EA) in CRC in vitro and in vivo. Our results showed that EA inhibited cell proliferation and migration in the CRC cell lines SW480 and HCT116. Moreover, EA significantly suppressed the growth of transplanted colorectal tumors in nude mice. Interestingly, high-throughput tandem mass tag (TMT)-based quantitative proteomics indicated that EA mainly targets tumor mitochondria and attenuates the translation of 54 mitochondrial ribosome proteins, many of which are discovered significantly upregulated in clinical CRC patients. More interestingly, EA at a low concentration (lower than 15 μg/ml) repressed the cell cycle by downregulating CDK1, CDK6, and CDC20, whereas at a high concentration (higher than 15 μg/ml), caused a non-apoptotic cell death—ferroptosis via downregulating ferritin (FTL) and upregulating transferrin (TF), ferroxidase (CP), and acyl-CoA synthetase long-chain family member 4 (ACSL4). This is the first report on the panoramic molecular mechanism of EA against CRC, which would make great contributions to developing a novel drug for colorectal cancer therapy.

LncRNA LINC00152 promotes oral squamous cell carcinoma growth via enhancing Upstream Transcription Factor 1 mediated Mitochondrial Ribosomal Protein L52 transcription.

LINC00152 (long intergenic non-protein coding RNA 152) was identified as an oncogenic lncRNA in multiple cancers. In the current study, we aimed to explore the transcriptional profile of LINC00152 in oral squamous cell carcinoma (OSCC) and its regulations at the transcriptional level. Bioinformatic analysis was performed by extracting the OSCC subset from The Cancer Genome Atlas (TCGA)-Head and Neck Squamous Cell Carcinoma (HNSC). LINC00152subcellular localization and its interacting transcriptional factors (TFs) were explored. Dual-luciferase assay and ChIP-qPCR were applied to study transcriptional regulation. In vitro and in vivo tumor cell growth models were used for functional assays. NR_024206.2 was the dominant isoform that accounts for 80% of all transcripts of LINC00152. LINC00152 upregulation was associated with unfavorable survival of patients with OSCC. LINC00152knockdown significantly impaired OSCC cell growth in vitro and in vivo. RNA FISH assay confirmed nuclear and cytoplasmic distribution of LINC00152. It physically interacted with Upstream Transcription Factor 1 (USF1), a common transcription factor in mammalian cells. USF1 could bind to the promoter region of MRPL52 (Mitochondrial Ribosomal Protein L52) and activate its transcription. LINC00152 could enhance the binding, thereby indirectly elevating MRPL52 expression. USF1 or MRPL52knockdown slowed the proliferation of OSCC cells and partly canceled LINC00152-mediated growth-promoting effects. This study revealed a novel LINC00152-USF1/MRPL52 axis promoting OSCC tumor growth.

Integrated proteome and phosphoproteome analysis of interscapular brown adipose and subcutaneous white adipose tissues upon high fat diet feeding in mouse

Thermogenesis is a promising approach to limit weight gain in response to excess nutrition. In contrast to cold-induced thermogenesis, the molecular and cellular mechanisms of diet-induced thermogenesis (DIT) have not been fully characterized. Here, we explored the response of brown adipose tissue (BAT) and subcutaneous white adipose tissue (sWAT) to high fat diet (HFD) using proteome and phosphoproteome analysis. We observed that after HFD, Uncoupling protein 1 (UCP1) and its phosphorylation were only increased in BAT. Furthermore, proteins involved in fatty acid oxidation, tricarboxylic acid cycle, and oxidative phosphorylation were also upregulated in BAT. Nevertheless, most metabolic related proteins were downregulated in sWAT. We found that these metabolic changes accompanied with different variation of mitochondrial proteins between BAT and sWAT. After HFD, most mitochondrial proteins were decreased in sWAT, but not in BAT. This effect was correlated with decreased mitochondrial ribosomal proteins in sWAT. Finally, through phosphoproteomic analysis, we predicted the activities of kinases in HFD mice and observed that there were more kinases inactivated in sWAT. Finally, this dataset provides a valuable resource for molecular researchers in the fields of obesity and obesity-related disease. SignificanceThermogenesis is a promising approach to combat obesity in response to excess energy. Nevertheless, the molecular and cellular mechanisms of DIT have not been fully characterized. Herein, we employed mass spectrometry (MS)-based proteomics and phosphoproteomics to identify differentially regulated proteins and phosphosites in BAT and sWAT of mice fed with HFD. This study unveils the differential regulatory networks of HFD in BAT and sWAT, which provides reference omics data to future researchers.

Development and Validation of a DNA Methylation-related Classifier of Circulating Tumour Cells to Predict Prognosis and to provide a therapeutic strategy in Lung Adenocarcinoma.

Background: A significant factor influencing the prognosis of lung adenocarcinoma (LUAD) is tumor metastasis. Studies have shown that abnormal DNA methylation in circulating tumor cells (CTCs) is associated with tumour metastasis. Based on the genes expressed in CTCs that play an important role in DNA methylation, we hope to build a risk model to predict prognosis and provide a therapeutic strategy in LUAD.Methods: The CTC sequencing data for LUAD were obtained from GSE74639, which contains 10 CTC samples and 6 primary tumour samples. To carefully assess the clinical value, functional status, involvement of the tumor microenvironment (TME) based on the risk model, and genetic variants based on based on data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO), a reliable risk model was successfully built.Results: Three differentially methylated genes (DMGs) of CTCs for LUAD, including mitochondrial ribosomal protein L51 (MRPL51), STE20-like kinase (SLK), and protein regulator of cytokinesis 1(PRC1), were effectively used to construct a risk model. Both the training and validation cohorts' stability and accuracy of the risk model were evaluated. Each patient in the TCGA-LUAD cohort received a risk score, and based on the median score, they were divided into high- and low-risk groups. The tumors in the high-risk group in this study were classified as "cold" and immunosuppressed, which may be linked to a poor prognosis. The tumors in the low-risk group, however, were deemed "hot" and had immune hyperfunction linked to a positive prognosis. Additionally, patients in the low-risk group showed greater sensitivity to immunotherapy than those in the high-risk group.Conclusions: Based on DMGs of CTCs from LUAD, we successfully developed a predictive risk model and discovered differences in biological function, TME, genetic variation, and clinical outcomes between those at high and low risk group.