Mitochondrial Ribosomal Protein Research Articles

Molecular pathways in mitochondrial disorders due to a defective mitochondrial protein synthesis

Mitochondria play a central role in cellular metabolism producing the necessary ATP through oxidative phosphorylation. As a remnant of their prokaryotic past, mitochondria contain their own genome, which encodes 13 subunits of the oxidative phosphorylation system, as well as the tRNAs and rRNAs necessary for their translation in the organelle. Mitochondrial protein synthesis depends on the import of a vast array of nuclear-encoded proteins including the mitochondrial ribosome protein components, translation factors, aminoacyl-tRNA synthetases or assembly factors among others. Cryo-EM studies have improved our understanding of the composition of the mitochondrial ribosome and the factors required for mitochondrial protein synthesis and the advances in next-generation sequencing techniques have allowed for the identification of a growing number of genes involved in mitochondrial pathologies with a defective translation. These disorders are often multisystemic, affecting those tissues with a higher energy demand, and often present with neurodegenerative phenotypes. In this article, we review the known proteins required for mitochondrial translation, the disorders that derive from a defective mitochondrial protein synthesis and the animal models that have been established for their study.

Advances in Mendelian randomization studies on autism spectrum disorder

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder with onset in infancy or early childhood. Mendelian randomization (MR) is a statistical method used to infer causal relationships between exposures and outcomes. This article summarizes MR studies related to ASD. Existing research supports a causal relationship between maternal inflammatory bowel disease in children with ASD, parental education levels, screen time exposure, obesity, insomnia, serum transferrin, decreased blood selenium, abnormal signals in brain functional MRI, interleukin-6, phosphodiesterase 2A, mitogen-activated protein kinase kinase 3, mitochondrial ribosomal protein L33, serotonin, and ASD. However, it does not support a causal relationship between parental rheumatoid arthritis, systemic lupus erythematosus, neonatal jaundice in children with ASD, cytomegalovirus infection, asthma, oral ulcers, vitamin D levels, and ASD. This article reviews the etiological factors related to ASD and MR studies, aiming to explore and deepen the understanding of the pathophysiology of ASD. It provides strong statistical support for the prevention, diagnosis, and treatment of ASD, and offers new methods and strategies for the etiological analysis of complex traits.

BmATAD3A mediates mitochondrial ribosomal protein expression to maintain the mitochondrial energy metabolism of the silkworm, Bombyx mori.

ATAD3A is a mitochondrial membrane protein belonging to the ATPase family that contains the AAA+ domain. It is widely involved in mitochondrial metabolism, protein transport, cell growth, development and other important life processes. It has previously been reported that the deletion of ATAD3A causes growth and development defects in humans, mice and Caenorhabditis elegans. To delve into the mechanism underlying ATAD3A defects and their impact on development, we constructed a Bombyx mori ATAD3A (BmATAD3A) defect model in silkworm larvae. We aim to offer a reference for understanding ATAD3A genetic defects and elucidating the molecular regulatory mechanisms. The results showed that knockout of the BmATAD3A gene significantly affected the weight, survival rate, ATPase production and mitochondrial metabolism of individuals after 24h of incubation. Combined metabolomics and transcriptomics analysis further demonstrated that BmATAD3A knockout inhibits amino acid biosynthesis through the regulation of mitochondrial ribosomal protein expression. Simultaneously, our findings indicate that BmATAD3A knockout impeded mitochondrial activity and ATPase synthesis and suppressed the mitochondrial oxidative phosphorylation pathway through B.mori mitochondrial ribosomal protein L11 (BmmRpL11). These results provide novel insights into the molecular mechanisms involved in the inhibition of development caused by ATAD3A deficiency, offering a potential direction for targeted therapy in diseases associated with abnormal ATAD3A expression.

LncRNA MRPL39 inhibits cell proliferation and migration by regulating miR-130/TSC1 axis in non-small cell lung cancer.

Currently, the effect of miR-130 on non-small cell lung cancer (NSCLC) remains controversial. In this study, the expression of miR-130 and lncRNA MRPL39 in tumor and non-tumor tissues of NSCLC patients was examined using real-time PCR (RT-PCR) and correlated with the prognosis of NSCLC. The phenotypic effects of miR-130 and MRPL39 on proliferation and migration of NSCLC cell line A549 cells were assessed through CCK-8 and Transwell assays with miR-130 mimic and MRPL39 (mitochondrial ribosomal protein L39) overexpressed plasmid transfection. StarBase/TargetScan analysis and dual-luciferase reporter gene assays were conducted to investigate the relationship between MRPL39, miR-130, and Tuberculosis sclerosis 1 (TSC1). MiR-130 was overexpressed, and MRPL39 was downregulated in NSCLC tissues and cells. Inhibition of miR-130 expression and overexpression of MRPL39 resulted in the inhibition of the viability and migration of A549 cells. MRPL39 is a potential upstream regulatory long non-coding RNA of miR-130, and its expression is negatively regulated by miR-130. TSC1 was identified as a target of miR-130, suppressing the antitumor effects of FGD5-AS1 silencing on GBM cells. After overexpression of MRPL39, the mRNA and protein levels of TSC1 in A549 cells significantly increased. However, after transfection with miR-130 mimic, the up-regulation of mRNA and protein was inhibited, leading to the suppression of cell proliferation and migration.

Mitochondrial ribosomal protein S24 is associated with immunosuppressive microenvironment and cold tumor in lung adenocarcinoma

ObjectiveMRPS24 (Mitochondrial Ribosomal Protein S24) belongs to the mitochondrial ribosomal protein family, which participates in the protein synthesis of the mitochondrion. However, the relationship of MRPS24 with lung adenocarcinoma (LUAD) remained unknown. We aimed to identify its immunological and functional mechanisms in LUAD. MethodsThe analysis of MRPS24 expression, clinical features, diagnosis, prognosis, function analysis, genetic alteration, copy number variations, methylation, and tumor microenvironment was investigated by the TCGA, UCSC Xena, GEO, HPA, GEPIA, cBioPortal, MethSurv, TIMER, TIMER2.0, and TISIDB databases. ResultsMRPS24 was found to be more abundant in LUAD tumor tissue than in normal tissue. High levels of MRPS24 expression were found to be an independent prognostic factor by multivariate analysis. Functional analysis revealed that MRPS24 expression was associated with the immune, cell cycle and methylation. MRPS24 methylation level was inversely linked with its expression (p < 0.001). Patients with low MRPS24 methylation had a worse prognosis than those with high methylation (p < 0.05). In addition, the result revealed that the MRPS24 expression was inversely linked to the immune cell infiltration in LUAD. Finally, the validations of the expression level, prognosis, and immune cell infiltration of MRPS24 were in accordance with our previous results. ConclusionsThis study systematically explored that MRPS24 expression was significantly correlated with prognosis, tumorigenesis, genetic alteration, copy number variations, methylation, and immune cell infiltration in LUAD. MRPS24 might be a potential immune-related biomarker in the development and treatment of LUAD, thereby acting as a promising predictor of immunotherapy response in LUAD.

Abstract 4296: Investigating metabolic sensitivity and organization of renal cell carcinoma: To enhance effective therapies and patient survival

Abstract Background: Renal cell carcinoma (RCC) is the 6th most common cancer in men and the 9th most common in women in the United States. RCC tissues are metabolically dysfunctional, often resulting from loss of von-Hippel Lindau (VHL) and consequent upregulation of hypoxia-inducible factor (HIF1/2). HIF1a is a transcription factor that upregulates glycolytic gene expression. Current therapies, including immune checkpoint inhibitors and tyrosine kinase inhibitors, have shown improvements in the outcomes of RCC patients, however, there are no biomarkers clinically available to predict the efficacy of these drugs. While genomic data and transcriptomic signatures have been investigated, they are not used in clinical practice due to lack of prospective validation. Understanding additional pathways pertaining to metabolic features of RCC, is critical. Our bulk gene expression analysis of primary RCC samples from TCGA-KIRC show a predominance of genes expressing mitochondrial ribosomal proteins and electron transport chain in tumors that relapsed early after nephrectomy, warranting further validation. Aim: We aim to investigate changes in metabolic adaptation in RCC cells from kidney cancer cell lines (VHL mutant) and primary cells from human-matched primary and metastatic kidney cancer sites. Using a novel biosensor-based technique developed in the Albeck lab, we aim to identify the dependence of live RCC cells on specific metabolic pathways and test the effect of standard-of-care (SOC) pathway inhibitors such as tyrosine kinase, HIF pathway, metabolic pathway inhibitors impacting tumorigenicity. Methods: To examine functional metabolic behavior in single cells, we generated immortalized cell lines (MCF10A, 184A1, FL83B and HBE-1) stably transfected with biosensors for the activity of AKT, glycolysis, mTOR and AMPK pathway. Data from Live-cells treated with metabolic inhibitors were collected and analyzed using our MATLAB-based pipeline. We have extended the analysis to RCC cell lines (786-O cell lines) and primary cells collected from nephrectomy specimens and matched metastatic biopsy samples, using current SOC pathway inhibitors (including sorafenib, sunitinib, belzutifan) and investigational metabolic drugs (metformin, IACS-010759, niclosamide). Results &amp; Conclusion: Our preliminary results show an association of high OX-PHOS with primary tumors from early relapse after nephrectomy than the relapsed late tumors. Our biosensor single cell data show that even genetically homogeneous cell populations can vary in their usage of OXPHOS and glycolysis to supply ATP for cell growth. Tumor gene expression profile coupled with biosensor data, will address mechanism behind this metabolic shift in RCC tumors in primary cells isolated from primary and metastatic sites. Our research will improve tumor prognosis and allow designing therapeutic trials. Citation Format: Madhura Patankar, John Albeck, Shuchi Gulati. Investigating metabolic sensitivity and organization of renal cell carcinoma: To enhance effective therapies and patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4296.

The zinc finger motif in the mitochondrial large ribosomal subunit protein bL36m is essential for optimal yeast mitoribosome assembly and function

Ribosomes across species contain subsets of zinc finger proteins that play structural roles by binding to rRNA. While the majority of these zinc fingers belong to the C2-C2 type, the large subunit protein L36 in bacteria and mitochondria exhibits an atypical C2-CH motif. To comprehend the contribution of each coordinating residue in S. cerevisiae bL36m to mitoribosome assembly and function, we engineered and characterized strains carrying single and double mutations in the zinc coordinating residues. Our findings reveal that although all four residues markedly influence protein stability, C to A mutations in C66 and/or C69 have a more pronounced effect compared to those at C82 and H88. Importantly, protein stability directly correlates with the assembly and function of the mitoribosome and the growth rate of yeast in respiratory conditions. Mass spectrometry analysis of large subunit particles indicates that strains deleted for bL36m or expressing mutant variants have defective assembly of the L7/L12 stalk base, limiting their functional competence. Furthermore, we employed a synthetic bL36m protein collection, including both wild-type and mutant proteins, to elucidate their ability to bind zinc. Our data indicate that mutations in C82 and, particularly, H88 allow for some zinc binding albeit inefficient or unstable, explaining the residual accumulation and activity in mitochondria of bL36m variants carrying mutations in these residues. In conclusion, stable zinc binding by bL36m is essential for optimal mitoribosome assembly and function. MS data are available via ProteomeXchange with identifierPXD046465.

Mitochondrial Ribosomal Protein MRPS15 Is a Component of Cytosolic Ribosomes and Regulates Translation in Stressed Cardiomyocytes.

Regulation of mRNA translation is a crucial step in controlling gene expression in stressed cells, impacting many pathologies, including heart ischemia. In recent years, ribosome heterogeneity has emerged as a key control mechanism driving the translation of subsets of mRNAs. In this study, we investigated variations in ribosome composition in human cardiomyocytes subjected to endoplasmic reticulum stress induced by tunicamycin treatment. Our findings demonstrate that this stress inhibits global translation in cardiomyocytes while activating internal ribosome entry site (IRES)-dependent translation. Analysis of translating ribosome composition in stressed and unstressed cardiomyocytes was conducted using mass spectrometry. We observed no significant changes in ribosomal protein composition, but several mitochondrial ribosomal proteins (MRPs) were identified in cytosolic polysomes, showing drastic variations between stressed and unstressed cells. The most notable increase in polysomes of stressed cells was observed in MRPS15. Its interaction with ribosomal proteins was confirmed by proximity ligation assay (PLA) and immunoprecipitation, suggesting its intrinsic role as a ribosomal component during stress. Knock-down or overexpression experiments of MRPS15 revealed its role as an activator of IRES-dependent translation. Furthermore, polysome profiling after immunoprecipitation with anti-MRPS15 antibody revealed that the "MRPS15 ribosome" is specialized in translating mRNAs involved in the unfolded protein response.

MRPL21 promotes HCC proliferation through TP53 mutation-induced apoptotic resistance

Background and AimsThe specific mechanisms underlying the inhibition of hepatocellular carcinoma (HCC) proliferation and metastasis by mitochondrial apoptosis are not yet fully understood. However, it plays a vital role in suppressing HCC's ability to proliferate and spread. The involvement of MRPL21, a member within the family of mitochondrial ribosomal proteins (MRPs), is well-documented in both cellular apoptosis and energy metabolism. This study aims to explore and unravel the underlying mechanisms through which MRPL21 contributes to mitochondrial apoptosis and resistance against apoptosis in HCC. MethodsTo evaluate the level of MRPL21 expression at the gene and protein expression levels, analysis was performed on human liver samples and blood using techniques for quantification. A knockdown plasmid targeting MRPL21 was constructed to investigate its impact on the growth and apoptosis of hepatocellular carcinoma (HCC). To evaluate the impact of MRPL21 knockdown on hepatocellular carcinoma (HCC) cell proliferation and apoptosis, various assays were performed including CCK-8 assays, flow cytometry analysis, detection of reactive oxygen species (ROS), and assessment of mitochondrial membrane potential (MMP). Furthermore, the role of MRPL21 in TP53 mutation was examined using Nutlin-3. ResultsIn HCC tissues and blood samples, an upregulation of MRPL21 expression was observed when compared to samples obtained from healthy individuals, and it is correlated with a poor prognosis for HCC. Silencing MRPL21 can effectively suppress Hep3B and HCCLM3 cells proliferation by modulating the mitochondrial membrane potential, it triggers the generation of reactive oxygen species (ROS), thereby leading to G0/G1 cell cycle arrest and initiation of early apoptosis. Furthermore, by inhibiting P53 activity, Nutlin-3 treatment can enhance MRPL21-deficiency-mediated apoptosis in Hep3B and HCCLM3 cells. ConclusionThrough its influence on TP53 mutation, MRPL21 promotes HCC proliferation and progression while conferring resistance to apoptosis. These findings suggest that MRPL21 holds promise as a valuable biomarker for the treatment of HCC.

New insights into the role of mitochondrial metabolic dysregulation and immune infiltration in septic cardiomyopathy by integrated bioinformatics analysis and experimental validation

BackgroundSeptic cardiomyopathy (SCM), a common cardiovascular comorbidity of sepsis, has emerged among the leading causes of death in patients with sepsis. SCM’s pathogenesis is strongly affected by mitochondrial metabolic dysregulation and immune infiltration disorder. However, the specific mechanisms and their intricate interactions in SCM remain unclear. This study employed bioinformatics analysis and drug discovery approaches to identify the regulatory molecules, distinct functions, and underlying interactions of mitochondrial metabolism and immune microenvironment, along with potential interventional strategies in SCM.MethodsGSE79962, GSE171546, and GSE167363 datasets were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and module genes were identified using Limma and Weighted Correlation Network Analysis (WGCNA), followed by functional enrichment analysis. Machine learning algorithms, including support vector machine–recursive feature elimination (SVM–RFE), least absolute shrinkage and selection operator (LASSO) regression, and random forest, were used to screen mitochondria-related hub genes for early diagnosis of SCM. Subsequently, a nomogram was developed based on six hub genes. The immunological landscape was evaluated by single-sample gene set enrichment analysis (ssGSEA). We also explored the expression pattern of hub genes and distribution of mitochondria/inflammation-related pathways in UMAP plots of single-cell dataset. Potential drugs were explored using the Drug Signatures Database (DSigDB). In vivo and in vitro experiments were performed to validate the pathogenetic mechanism of SCM and the therapeutic efficacy of candidate drugs.ResultsSix hub mitochondria-related DEGs [MitoDEGs; translocase of inner mitochondrial membrane domain-containing 1 (TIMMDC1), mitochondrial ribosomal protein S31 (MRPS31), F-box only protein 7 (FBXO7), phosphatidylglycerophosphate synthase 1 (PGS1), LYR motif containing 7 (LYRM7), and mitochondrial chaperone BCS1 (BCS1L)] were identified. The diagnostic nomogram model based on the six hub genes demonstrated high reliability and validity in both the training and validation sets. The immunological microenvironment differed between SCM and control groups. The Spearman correlation analysis revealed that hub MitoDEGs were significantly associated with the infiltration of immune cells. Upregulated hub genes showed remarkably high expression in the naive/memory B cell, CD14+ monocyte, and plasma cell subgroup, evidenced by the feature plot. The distribution of mitochondria/inflammation-related pathways varied across subgroups among control and SCM individuals. Metformin was predicted to be the most promising drug with the highest combined score. Its efficacy in restoring mitochondrial function and suppressing inflammatory responses has also been validated.ConclusionsThis study presents a comprehensive mitochondrial metabolism and immune infiltration landscape in SCM, providing a potential novel direction for the pathogenesis and medical intervention of SCM.

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production.

Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS-based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III, and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits-including NDUFA8, NDUFB4, and NDUFS8-decreased complex I activity, mitochondria-derived ATP, and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

Dysregulated Ribosome Biogenesis Is a Targetable Vulnerability in Triple-Negative Breast Cancer: MRPS27 as a Key Mediator of the Stemness-inhibitory Effect of Lovastatin.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited effective therapeutic options readily available. We have previously demonstrated that lovastatin, an FDA-approved lipid-lowering drug, selectively inhibits the stemness properties of TNBC. However, the intracellular targets of lovastatin in TNBC remain largely unknown. Here, we unexpectedly uncovered ribosome biogenesis as the predominant pathway targeted by lovastatin in TNBC. Lovastatin induced the translocation of ribosome biogenesis-related proteins including nucleophosmin (NPM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and the ribosomal protein RPL3. Lovastatin also suppressed the transcript levels of rRNAs and increased the nuclear protein level and transcriptional activity of p53, a master mediator of nucleolar stress. A prognostic model generated from 10 ribosome biogenesis-related genes showed outstanding performance in predicting the survival of TNBC patients. Mitochondrial ribosomal protein S27 (MRPS27), the top-ranked risky model gene, was highly expressed and correlated with tumor stage and lymph node involvement in TNBC. Mechanistically, MRPS27 knockdown inhibited the stemness properties and the malignant phenotypes of TNBC. Overexpression of MRPS27 attenuated the stemness-inhibitory effect of lovastatin in TNBC cells. Our findings reveal that dysregulated ribosome biogenesis is a targetable vulnerability and targeting MRPS27 could be a novel therapeutic strategy for TNBC patients.

The role of lysine acetylation in the function of mitochondrial ribosomal protein L12.

Mitochondria play a central role in energy production and cellular metabolism. Mitochondria contain their own small genome (mitochondrial DNA, mtDNA) that carries the genetic instructions for proteins required for ATP synthesis. The mitochondrial proteome, including the mitochondrial transcriptional machinery, is subject to post-translational modifications (PTMs), including acetylation and phosphorylation. We set out to determine whether PTMs of proteins associated with mtDNA may provide a potential mechanism for the regulation of mitochondrial gene expression. Here, we focus on mitochondrial ribosomal protein L12 (MRPL12), which is thought to stabilize mitochondrial RNA polymerase (POLRMT) and promote transcription. Numerous acetylation sites of MRPL12 were identified by mass spectrometry. We employed amino acid mimics of the acetylated (lysine to glutamine mutants) and deacetylated (lysine to arginine mutants) versions of MRPL12 to interrogate the role of lysine acetylation in transcription initiation in vitro and mitochondrial gene expression in HeLa cells. MRPL12 acetyl and deacetyl protein mimics were purified and assessed for their ability to impact mtDNA promoter binding of POLRMT. We analyzed mtDNA content and mitochondrial transcript levels in HeLa cells upon overexpression of acetyl and deacetyl mimics of MRPL12. Our results suggest that MRPL12 single-site acetyl mimics do not change the mtDNA promoter binding ability of POLRMT or mtDNA content in HeLa cells. Individual acetyl mimics may have modest effects on mitochondrial transcript levels. We found that the mitochondrial deacetylase, Sirtuin 3, is capable of deacetylating MRPL12 in vitro, suggesting a potential role for dynamic acetylation controlling MRPL12 function in a role outside of the regulation of gene expression.

LARP4 is an RNA-binding protein that binds nuclear-encoded mitochondrial mRNAs to promote mitochondrial function.

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.

Mitochondrial ribosomal protein L12 potentiates hepatocellular carcinoma by regulating mitochondrial biogenesis and metabolic reprogramming

BackgroundMitochondrial dysfunction and metabolic reprogramming are key features of hepatocellular carcinoma (HCC). Despite its significance, the precise underlying mechanism behind these processes has not been fully elucidated. The latest investigations, along with our previous discoveries, have substantiated the significant role of mitochondrial ribosomal protein L12 (MRPL12), a newly identified gene involved in mitochondrial transcription regulation, in the modulation of mitochondrial metabolism. Nevertheless, the role of MRPL12 in tumorigenesis has yet to be investigated. MethodsThe expression of MRPL12 in HCC was assessed using an online database. Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry (IHC) were employed to determine the expression of MRPL12 in HCC tissues, patient-derived organoid (PDO), and cell lines. The correlation between MRPL12 expression and clinicopathological features, as well as prognosis, was examined using tissue microarray analysis. An in vivo subcutaneous tumor xenograft model, gene knockdown or overexpression assay, chromatin immunoprecipitation (ChIP) assay, Seahorse XF96 assay, and cell function assay were employed to investigate the biological function and potential molecular mechanism of MRPL12 in HCC. ResultsA significant upregulation of MRPL12 was observed in HCC cells, PDO and patient tissues, which correlated with advanced tumor stage, higher grade and poor prognosis. MRPL12 overexpression promoted cell proliferation, migration, and invasion in vitro, as well as tumorigenicity in vivo, whereas MRPL12 knockdown showed the opposite effect. MRPL12 knockdown also inhibited the capacity of organoids proliferation capacity. Furthermore, MRPL12 was found to be crucial for maintaining mitochondrial homeostasis. Both gain and loss-of-function experiments targeting MRPL12 in HCC cells altered oxidative phosphorylation (OXPHOS) and mitochondrial DNA content. Notably, suppression of OXPHOS effectively mitigates the tumor-promoting effect attributed to MRPL12 overexpression, implying the involvement of MRPL12 in HCC through the modulation of mitochondrial metabolism. Besides, Yin Yang 1 (YY1) was identified as a transcription factor responsible for regulating MRPL12, while the PI3K/mTOR pathway was found to act as an upstream regulator of YY1. MRPL12 knockdown attenuated the YY1 overexpression or PI3K/mTOR activation-induced malignant phenotype in HCC cells. ConclusionOur findings provide compelling evidence that MRPL12 is implicated in driving the malignant phenotype of HCC via regulating mitochondrial metabolism. Moreover, the aberrant expression of MRPL12 in HCC is mediated by the upstream PI3K/mTOR/YY1 pathway. These results highlight the potential of targeting MRPL12 as a promising therapeutic strategy for the treatment of HCC.

MRPL48 is a novel prognostic and predictive biomarker of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most prevalent forms of cancer and poses a threat to the health and survival of humans. Mitochondrial ribosomal protein L48 (MRPL48) belongs to the mitochondrial ribosomal protein family, which participates in energy production. Studies have shown that MRPL48 can predict osteosarcoma incidence and prognosis, as well as promotes colorectal cancer progression. However, the role of MRPL48 in HCC remains unknown.MethodsTCGA, GEO, HCCDB, CPTAC, SMART, UALCAN, Kaplan–Meier plotter, cBioPortal, and MethSurv were performed for bioinformatics purposes. Quantitative RT-PCR, immunoblotting, and functional studies were conducted to validate the methodology in vitro.ResultsMRPL48 was greatly overexpressed in HCC tissues, compared with healthy tissue, which was subsequently demonstrated in vitro as well. The survival and regression analyses showed that MRPL48 expression is of significant clinical prognostic value in HCC. The ROC curve and nomogram analysis indicated that MRPL48 is a powerful predictor of HCC. MRPL48 methylation was adversely associated with the expression of MRPL48, and patients with a low level of methylation had poorer overall survival than those with a high level of methylation. GSEA showed that the expression of the MRPL48 was correlated with Resolution of Sister Chromatid Cohesion, Mitotic Prometaphase, Retinoblastoma Gene in Cancer, RHO Gtpases Activate Formins, Mitotic Metaphase and Anaphase, and Cell Cycle Checkpoints. An analysis of immune cell infiltration showed a significant association between MRPL48 and immune cell infiltration subsets, which impacted the survival of HCC patients. Additionally, MRPL48 knockdown reduced HCC cell proliferation, migration, and invasion in vitro.ConclusionsWe demonstrated that MRPL48 expression may be associated with HCC development and prognosis. These findings may open up new research directions and opportunities for the development of HCC treatments.

In silico analyses of pan-squamous cell carcinoma unveiled the immunological implications of MRPL13, which had previously been under-recognized

The involvement of the mitochondrial ribosomal protein 13 (MRPL13) gene in the development of adenocarcinoma has been previously reported. However, the clinicopathological significance of MRPL13 in squamous cell carcinoma (SCC) remains poorly understood. To gain insight into the clinicopathological and immunological implications of MRPL13 expression in SCC, we conducted a bioinformatic analysis utilizing various available databases, including TIMER 2.0, Xiantao academic tool and TISIDB, attempting to evaluate the abnormal expression, prognosis and immunological correlation of MRPL13 in the pan-SCC setting. Subsequently, we conducted experimental verification using an esophageal squamous cell carcinoma (ESCC) tissue array subjected to multiplexed immunofluorescent (mIF) staining. The ESCC tissue array we used consists of 93 dots of ESCC and 86 dots of matched adjacent normal tissues (ANT). Data from in silico analyses showed that MRPL13 mRNA is significantly up-regulated and correlated with infiltration of CD8+ T cells in pan-SCC. However, in silico analyses did not support the prognostic role of MRPL13 in SCC. Consistently, data from the ESCC tissue array showed that MRPL13 was remarkably elevated in ESCC tissues relative to ANT in stroma, which was controlled by pan-cytokeratin (pan-CK) staining. In the epithelia, no significant difference was identified between ESCC and ANT. Furthermore, MRPL13 expression markedly correlated with the infiltration of CD8+ T cells in the stromal region but not in the epithelial region. Prognostically, no significant association was observed between MRPL13 expression and overall survival, regardless of epithelial or stromal section. Through these pan-SCC analyses, we have expanded the understanding of MRPL13 previously reported, in particular, underscoring the immunological involvement of MRPL13 in the tumor microenvironment of SCC that has been under-recognized before, suggesting that MRPL13 may regulate the infiltration of CD8+ T cells into the SCC microenvironment.

Genetic, metabolic and clinical delineation of an MRPS23-associated mitochondrial disorder

MRPS23 is a nuclear gene encoding a mitochondrial ribosomal protein. A patient with a mitochondrial disorder was found to carry a variant in MRPS23. More cases are necessary to establish MRPS23 as a mitochondrial disease gene. Of 5134 exomes performed in our center, we identified five independent patients who had similar clinical manifestations and were homozygous for the same germline variant c.119C>T; p.P40L in MRPS23. Detailed clinical findings, mitochondrial enzyme activity assays from cultured skin fibroblasts, PCR-Sanger-sequencing, and variant age estimation were performed. Their available family members were also studied. Eight members homozygous for the MRPS23 p.P40L were identified. All were from Hmong hilltribe. Seven presented with alteration of consciousness and recurrent vomiting, while the eighth who was a younger brother of a proband was found pre-symptomatically. Patients showed delayed growth and development, hearing impairment, hypoglycemia, lactic acidosis, and liver dysfunction. In vitro assays of cultured fibroblasts showed combined respiratory chain complex deficiency with low activities of complexes I and IV. PCR-Sanger-sequencing confirmed the variant, which was estimated to have occurred 1550 years ago. These results establish the MRPS23-associated mitochondrial disorder inherited in an autosomal recessive pattern and provide insight into its clinical and metabolic features.

Screening and identification of key biomarkers associated with amyotrophic lateral sclerosis and depression using bioinformatics.

This study aims to identify common molecular biomarkers between amyotrophic lateral sclerosis (ALS) and depression using bioinformatics methods, in order to provide potential targets and new ideas and methods for the diagnosis and treatment of these diseases. Microarray datasets GSE139384, GSE35978 and GSE87610 were obtained from the Gene Expression Omnibus (GEO) database, and differentially expressed genes (DEGs) between ALS and depression were identified. After screening for overlapping DEGs, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Furthermore, a protein-protein interaction (PPI) network was constructed using the STRING database and Cytoscape software, and hub genes were identified. Finally, a network between miRNAs and hub genes was constructed using the NetworkAnalyst tool, and possible key miRNAs were predicted. A total of 357 genes have been identified as common DEGs between ALS and depression. GO and KEGG enrichment analyses of the 357 DEGs showed that they were mainly involved in cytoplasmic translation. Further analysis of the PPI network using Cytoscape and MCODE plugins identified 6 hub genes, including mitochondrial ribosomal protein S12 (MRPS12), poly(rC) binding protein 1 (PARP1), SNRNP200, PCBP1, small G protein signaling modulator 1 (SGSM1), and DNA methyltransferase 1 (DNMT1). Five possible target miRNAs, including miR-221-5p, miR-21-5p, miR-100-5p, miR-30b-5p, and miR-615-3p, were predicted by constructing a miRNA-gene network. This study used bioinformatics techniques to explore the potential association between ALS and depression, and identified potential biomarkers. These biomarkers may provide new ideas and methods for the early diagnosis, treatment, and monitoring of ALS and depression.

Abstract 17551: Mapping the Interactome of FLNC in Restrictive Cardiomyopathy

Introduction: Filamin C (FLNC) is an actin crosslinking protein that organizes structural components of the sarcomere and signal transduction complexes. Human mutations in FLNC have been linked to dilated, hypertrophic and restrictive cardiomyopathies. The mechanism connecting mutations in FLNC to sarcomeric impairment remains undefined. We recently reported the ability to model FLNC cardiomyopathy using induced pluripotent stem cell (iPSC) derived engineered heart tissue. Here, we employ a knock-in approach to epitope tag endogenous alleles in iPSC derived cardiomyocytes to explore FLNC genotype-interactome relationships using affinity purification mass spectrometry. Methods: An iPSC line was generated from a patient with restrictive cardiomyopathy caused by an insertion deletion mutation in the ROD2 domain FLNC (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp). CRISPR-Cas9 was used to establish a corrected cell line as a wild type control. 3X-Flag motif was introduced to the N terminus of FLNC using CRISPR-Cas9. iPSCs were then differentiated into cardiomyocytes using standard techniques. Affinity purification mass spectrometry (AP-MS) was performed using the Flag epitope. Interaction data was analyzed using Perseus software. Gene ontology (GO) enrichment analysis was performed using ToppFun. Results: 3X-Flag insertion was confirmed using genomic sequencing. AP-MS identified 281 high confidence interactors of mutant FLNC and 208 interactors of wild type FLNC (FDR 0.01, log2FC &gt;1.5) when compared to IgG controls. Interestingly, 161 proteins were shared between the two genotypes. These were enriched in mitochondrial ribosomal proteins and phosphorylase kinase subunits, potentially revealing an unknown role of FLNC in mitochondria translation and metabolism. Thirty-three proteins were significantly differentially bound to mutant FLNC protein, including tropomyosin 4, caldesmon 1, and filamin A (FDR &lt;0.05, log2FC&gt;1). GO analysis of these proteins revealed enrichment of actin cytoskeleton elements, muscle contraction, and muscle system processes. Conclusions: Human mutations in FLNC lead to disruption of key protein interactions, revealing a potential mechanism for disease pathogenesis in FLNC cardiomyopathy.