Mitochondrial Import Research Articles

Abstract 14145: The Zinc Transporter ZIP7 Plays a Critical Role in Ferrotposis Induced by Ischemia/Reperfusion in Mouse Hearts

Background: Although ferroptosis has been proposed to play a role in myocardial ischemia/reperfusion (I/R) injury, the exact molecular mechanism by which ferroptosis is induced by I/R remains unclear. Hypothesis: We tested the hypothesis that upregulation of the zinc transporter ZIP7 at reperfusion triggers ferroptosis by downregulating the mitochondrial GPX4 (mtGPX4) via the guanine-rich sequence-binding factor 1 (GRSF1) and increasing mitochondrial iron accumulation through inhibition of the mitochondrial iron importer degradation. Aims: To determine if ZIP7 plays a role in I/R-induced ferroptosis and to investigate the underlying molecular mechanism. Methods: Mouse hearts were subjected to I/R in vivo . The cardiac-specific Slc39a7 conditional knockout mice (ZIP7 cKO) were generated by adopting the CRISPR/Cas9 system. Adult mouse cardiomyocytes were isolated enzymatically. Ferroptosis was evaluated by detecting PTGS2, MDA, and BODIPY C11. Mitochondrial Fe 2+ was determined with Mito-FerroGreen fluorescent probe. Results: I/R upregulated ZIP7 expression and induced ferroptosis as indicated by the increases in PTGS2 mRNA expression (290.90 ± 52.78 % of control), MDA (152.10 ± 15.07 % of control) and BODIPY C11 (130.68 ± 5.52 % of control) levels, which was prevented by ZIP7 cKO. ZIP7 cKO also mitigated doxorubicin-induced ferroptosis. ZIP7 cKO upregulated mtGPX4 expression (123.40±3.26 % of I/R) and increased mitochondrial GRSF1 expression (150.90±8.51 % of I/R) at reperfusion. In addition, knockout of GRSF1 inhibited GPX4 expression (54.73±5.44 % of control). Moreover, ZIP7 cKO prevented the accumulation of Fe 2+ within mitochondria (44.15±2.46 % of I/R) and promoted the degradation of SLC25A37 (62.57±8.03% of I/R) and SLC25A28 (69.82±3.10 % of I/R) at reperfusion. Conclusions: Upregulation of ZIP7 contributes to the induction of ferroptosis at reperfusion presumably by reducing mtGPX4 expression via GRSF1and increasing Fe 2+ accumulation within mitochondria through inhibition of SLC25A37 and SLC25A28 degradation.

Interactions of amyloidogenic proteins with mitochondrial protein import machinery in aging-related neurodegenerative diseases.

Most mitochondrial proteins are targeted to the organelle by N-terminal mitochondrial targeting sequences (MTSs, or "presequences") that are recognized by the import machinery and subsequently cleaved to yield the mature protein. MTSs do not have conserved amino acid compositions, but share common physicochemical properties, including the ability to form amphipathic α-helical structures enriched with basic and hydrophobic residues on alternating faces. The lack of strict sequence conservation implies that some polypeptides can be mistargeted to mitochondria, especially under cellular stress. The pathogenic accumulation of proteins within mitochondria is implicated in many aging-related neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. Mechanistically, these diseases may originate in part from mitochondrial interactions with amyloid-β precursor protein (APP) or its cleavage product amyloid-β (Aβ), α-synuclein (α-syn), and mutant forms of huntingtin (mHtt), respectively, that are mediated in part through their associations with the mitochondrial protein import machinery. Emerging evidence suggests that these amyloidogenic proteins may present cryptic targeting signals that act as MTS mimetics and can be recognized by mitochondrial import receptors and transported into different mitochondrial compartments. Accumulation of these mistargeted proteins could overwhelm the import machinery and its associated quality control mechanisms, thereby contributing to neurological disease progression. Alternatively, the uptake of amyloidogenic proteins into mitochondria may be part of a protein quality control mechanism for clearance of cytotoxic proteins. Here we review the pathomechanisms of these diseases as they relate to mitochondrial protein import and effects on mitochondrial function, what features of APP/Aβ, α-syn and mHtt make them suitable substrates for the import machinery, and how this information can be leveraged for the development of therapeutic interventions.

The Clpx G298D Mutation in Clpx-EPP Reveals That Clpx Regulates the Mitochondria Enzymes in Erythroid Heme Synthesis Via Distinct Mechanisms

Terminally differentiating red cells synthesize large quantities of heme for hemoglobin production, requiring tight coordination between mitochondrial iron import and synthesis of photosensitive protoporphyrin IX (PPIX). PPIX causes erythropoietic porphyria from loss of function mutations in FECH, or gain of function mutations in ALAS2. We previously reported that a dominant heterozygous point mutation in human CLPX (G298D) was a novel cause of erythropoietic protoporphyria (Yien et al. 2017, PNAS). CLPX encodes a ubiquitous ATPase-dependent mitochondrial protein unfoldase and is required for tight regulation of the mitochondrial heme synthesis enzymes, ALAS 1 and 2, FECH and PPOX, as well as mitochondrial iron utilization. CLPX facilitates degradation of the ALAS enzymes, but paradoxically, is also required for activity of the PPOX and FECH enzymes. CLPX function is required for porphyrin synthesis and for the incorporation of iron into heme (Rondelli et al. 2021, JBC). Because the role of CLPX in heme regulation is complex and cell-specific, we sought to interrogate the mechanism by which the CLPX G298D mutant protein caused erythroid protoporphyria. To determine the requirement for CLPX for maximal heme synthesis, we labeled wild-type and Clpx -/- 3T3 murine fibroblasts and G1E-ER4 erythroid cells with 14C-aminolevulinate (ALA) and measured heme synthesis by liquid scintillation to ensure that equal amounts of labelled ALA were entering the heme synthesis pathway, uncoupling the effects of CLPX on the first and terminal steps of heme synthesis. We found that Clpx -/- cells synthesized significantly less 14C-ALA -labelled heme, indicating that CLPX is required for activation of the terminal heme synthesis enzymes in both erythroid and non-erythroid cells. Mass spectrometric quantification of FECH and PPOX protein and enzyme activity assays indicated that their protein levels and activities were decreased in Clpx -/- cells. Since CLPX ubiquitously regulates the mitochondrial steps of heme synthesis, we sought to determine why the porphyrin accumulation in CLPX -EPP predominantly occurred in erythroid cells and to interrogate the pathogenic mechanism of CLPX-EPP. To address this question, we knocked in the human G298D/mouse G299D mutation into murine erythroleukemia cells. The heterozygous mutant cells, denoted as WT/G299D, synthesized normal levels of heme. Further, differentiating WT/G299D cells synthesized significantly more porphyrin than wild-type cells, phenocopying the patient. In contrast, non-differentiating WT/G299D cells produced wild-type levels of porphyrin. ALAS2 was stabilized in the mutant expressing cells, likely accounting for the increase in porphyrin during erythroid differentiation. Iron supplementation was effective in decreasing photosensitivity and porphyrin levels in the patient (Ducamp et al., Hematologica 2021) as was predicted for porphyrias caused by increases in ALAS2 activity (Landefeld et al., Br. J. Hematol. 2016). Similarly, supplemental iron was also able to decrease porphyrin levels in WT/G299D cells to wild-type levels (Figure 1). In contrast to the WT/G299D cells, wild-type cells did not experience a decrease in porphyrin synthesis. We also did not observe increased heme synthesis with iron supplementation, indicating that iron, rather than promoting heme synthesis, is inhibiting an upstream step of porphyrin synthesis in WT/G299D cells. Secondly, co-immunoprecipitation assays indicated ATPase activity or ATP binding was dispensable for the interaction between CLPX and PPOX/FECH, likely accounting for the normal heme synthesis observed in CLPX G299D expressing cells. Lastly, the G299D mutant protein has decreased ALAS degradation activity, while retaining its ability to activate PPOX/FECH. This indicates that the CLPX protein is multifunctional in heme regulation. The control of heme synthesis by CLPX unveils a novel cause of protoporphyria and the erythroid specificity of the disease indicates that CLPX has erythroid-specific functions in heme regulation. Further, while iron promotes ALAS2 translation, iron supplementation also suppresses porphyrin synthesis in CLPX-EPP. We reveal an important erythroid-specific regulatory node where the mitochondrial protein homeostasis intersects with heme synthesis, offering important insights on the pathogenesis and treatment of porphyrias.

LPGAT1 controls MEGDEL syndrome by coupling phosphatidylglycerol remodeling with mitochondrial transport

Phosphatidylglycerol (PG) is a mitochondrial phospholipid required for mitochondrial cristae structure and cardiolipin synthesis. PG must be remodeled to its mature form at the endoplasmic reticulum (ER) after mitochondrial biosynthesis to achieve its biological functions. Defective PG remodeling causes MEGDEL (non-alcohol fatty liver disease and 3-methylglutaconic aciduria with deafness, encephalopathy, and Leigh-like) syndrome through poorly defined mechanisms. Here, we identify LPGAT1, an acyltransferase that catalyzes PG remodeling, as a candidate gene for MEGDEL syndrome. We show that PG remodeling by LPGAT1 at the ER is closely coordinated with mitochondrial transport through interaction with the prohibitin/TIMM14 mitochondrial import motor. Accordingly, ablation of LPGAT1 or TIMM14 not only causes aberrant fatty acyl compositions but also ER retention of newly remodeled PG, leading to profound loss in mitochondrial crista structure and respiration. Consequently, genetic deletion of the LPGAT1 in mice leads to cardinal features of MEGDEL syndrome, including 3-methylglutaconic aciduria, deafness, dilated cardiomyopathy, and premature death, which are highly reminiscent of those caused by TIMM14 mutations in humans.

Investigation of the Molecular Mechanisms of Mitochondrial Import and Maturation of Human Peroxiredoxins Prdx3 and Prdx5

Investigation of the Molecular Mechanisms of Mitochondrial Import and Maturation of Human Peroxiredoxins Prdx3 and Prdx5

Decoding mitochondrial-nuclear (epi)genome interactions: the emerging role of ncRNAs.

Bidirectional communication between the mitochondria and the nucleus is required for several physiological processes, and the nuclear epigenome is a key mediator of this relationship. ncRNAs are an emerging area of discussion for their roles in cellular function and regulation. In this review, we highlight the role of mitochondrial-encoded ncRNAs as mediators of communication between the mitochondria and the nuclear genome. We focus primarily on retrograde signaling, a process in which the mitochondrion relays ncRNAs to translate environmental stress signals to changes in nuclear gene expression, with implications on stress responses that may include disease(s). Other biological roles of mitochondrial-encoded ncRNAs, such as mitochondrial import of proteins and regulation of cell signaling, will also be discussed.

New roles of LPGAT1: From mitochondrial import of phosphatidylglycerol to MEGDEL disease

Dysregulation of mitochondrial lipidome is associated with several human pathologies. Sun etal.1 showthat LPGAT1 cooperates with TIMM14 to regulate phosphatidylglycerol transport from the endoplasmic reticulum to the mitochondria, and uncover the involvement of LPGAT1 deficiency in MEGDEL syndrome.

Reduced Mitochondrial Protein Translation Promotes Cardiomyocyte Proliferation and Heart Regeneration.

The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation. We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed. We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5+/-/Atf4+/-), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell-derived cardiomyocytes. These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.

Mitochondrial Translocase TOMM22 Is Overexpressed in Pancreatic Cancer and Promotes Aggressive Growth by Modulating Mitochondrial Protein Import and Function.

TOMM22 bears potential for early diagnostic/prognostic biomarker development and therapeutic targeting for better management of patients with pancreatic cancer.

Combined Oxidative Phosphorylation Deficiency Type-13 with Perinatal Presentation: A Case Report.

Polynucleotide phosphorylase is involved in RNA processing in mitochondria. Biallelic variants in PNPT1 cause mitochondrial RNA import protein deficiency and heterogeneous clinical manifestations. The patiest was the first child of remote consanguineous parents, born at 35 weeks by caesarean section due to fetal growth restriction. Apgar index was 9/10/10. Birth weight, length and head circumference were at 3rd, <3rd and 10th percentiles, respectively. In the first hours of life, respiratory distress, hypoglycaemia and seizures ensued. She started invasive mechanic ventilation, phenobarbital and was transferred to ICU. Physical examination showed minor facial dysmorphisms, brief eye-opening, hypotonia and hyporeflexia. Electroencephalogram showed immature pattern and multifocal paroxysmal activity. MRI at D8 of life showed severe reduced brain volume. Normal aminoacid screen was also observed. Expanded newborn screening was negative. Mitochondrial organic aciduria was seen. WES showed a homozygotic likely pathogenic variant in the PNPT1 gene. MRI at 6-months showed brain atrophy, thin corpus callosum, reduced brainstem volume. Bilateral and symmetrical lesions in globi pallidi, compatible with Leigh síndrome were observed. Currently, at 14 months, no neurodevelopment progress, dystonia, visual deficit, sensorineural deafness, hypertrophic cardiomyopathy and microcephaly are observed. The early and severe Leigh-like presentation of our patient expands the phenotype spectrum of this disease. As far as we know, this is the first reported case of PNPT1 mutation with onset in the perinatal period. Moreover, hypertrophic cardiomyopathy has not yet been described in association with mutation of the PNPT1 gene. WES was the key for early diagnosis in this patient. It should be done in all children with severe clinical presentation of unknown origin.

ATP synthase-associated coiled-coil-helix-coiled-coil-helix (CHCH) domain-containing proteins are critical for mitochondrial function in Toxoplasma gondii.

Members of the coiled-coil-helix-coiled-coil-helix (CHCH) domain protein family are transported into the mitochondrial intermembrane space, where they play important roles in the biogenesis and function of the organelle. Unexpectedly, the ATP synthase of the apicomplexan Toxoplasma gondii harbors CHCH domain-containing subunits of unknown function. As no other ATP synthase studied to date contains this class of proteins, characterizing their function will be of broad interest to the fields of molecular parasitology and mitochondrial evolution. Here, we demonstrate that that two T. gondii ATP synthase subunits containing CHCH domains are required for parasite survival and for stability and function of the ATP synthase. We also show that knockdown disrupts multiple aspects of the mitochondrial morphology of T. gondii and that mutation of key residues in the CHCH domains caused mis-localization of the proteins. This work provides insight into the unique features of the apicomplexan ATP synthase, which could help to develop therapeutic interventions against this parasite and other apicomplexans, such as the malaria-causing parasite Plasmodium falciparum.

Yeast mitochondria can process de novo designed β-barrel proteins.

Mitochondrial outer membrane β-barrel proteins are encoded in the nucleus, translated in the cytosol and then targeted to and imported into the respective organelles. Detailed studies have uncovered the mechanisms involved in the import of these proteins and identified the targeting signals and the cytosolic factors that govern their proper biogenesis. Recently, de novo designed eight-stranded β-barrel proteins (Tmb2.3 and Tmb2.17) were shown to fold and assemble into lipid membranes. To better understand the general aspects of the biogenesis of β-barrel proteins, we investigated the fate of these artificial proteins upon their expression in yeast cells. We demonstrate that although these proteins are de novo designed and are not related to bona fide mitochondrial β-barrel proteins, they were targeted to mitochondria and integrated into the organelle outer membrane. We further studied whether this integration requires components of the yeast mitochondrial import machinery like Tom20, Tom70, Tob55/Sam50 and Mas37/Sam37. Whereas it seems that none of the import receptors was required for the biogenesis of the artificial β-barrel proteins, we observed a strong dependency on the TOB/SAM complex. Collectively, our findings demonstrate that the mitochondrial outer membrane is the preferential location in yeast cells for any membrane-embedded β-barrel protein.

Mitochondrial DNA breaks activate an integrated stress response to reestablish homeostasis

Mitochondrial DNA double-strand breaks (mtDSBs) lead to the degradation of circular genomes and a reduction in copy number; yet, the cellular response in human cells remains elusive. Here, using mitochondrial-targeted restriction enzymes, we show that a subset of cells with mtDSBs exhibited defective mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy confirmed the altered mitochondrial membrane and cristae ultrastructure. Intriguingly, mtDSBs triggered the integrated stress response (ISR) via the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by DELE1 and heme-regulated eIF2α kinase (HRI). When ISR was inhibited, the cells experienced intensified mitochondrial defects and slower mtDNA recovery post-breakage. Lastly, through proteomics, we identified ATAD3A-a membrane-bound protein interacting with nucleoids-as potentially pivotal in relaying signals from impaired genomes to the inner mitochondrial membrane. In summary, our study delineates the cascade connecting damaged mitochondrial genomes to the cytoplasm and highlights the significance of the ISR in maintaining mitochondrial homeostasis amid genome instability.

Molecular dynamics simulation reveals DNA-specific recognition mechanism via c-Myb in pseudo-palindromic consensus of mim-1 promoter.

This study aims to gain insight into the DNA-specific recognition mechanism of c-Myb transcription factor during the regulation of cell early differentiation and proliferation. Therefore, we chose the chicken myeloid gene, mitochondrial import protein 1 (mim-1), as a target to study the binding specificity between potential dual-Myb-binding sites. The c-Myb-binding site in mim-1 is a pseudo-palindromic sequence AACGGTT, which contains two AACNG consensuses. Simulation studies in different biological scenarios revealed that c-Myb binding with mim-1 in the forward strand (complex F) ismore stable than that inthereverse strand (complex R). The principal component analysis (PCA) dynamics trajectory analyses suggested an opening motion of the recognition helices of R2 and R3 (R2R3), resulting in the dissociation of DNA from c-Myb in complex R at 330 K, triggered by the reduced electrostatic potential on the surface of R2R3. Furthermore, the DNA confirmation and hydrogen-bond interaction analyses indicated that the major groove width of DNA increased in complex R, which affected on the hydrogen-bond formation ability between R2R3 and DNA, and directly resulted in the dissociation of DNA from R2R3. The steered molecular dynamics (SMD) simulation studies also suggested that the electrostatic potential, major groove width, and hydrogen bonds made major contribution to the DNA‍-specific recognition. In vitro trials confirmed the simulation results that c-Myb specifically bound to mim-1 in the forward strand. This study indicates that the three-dimensional (3D) structure features play an important role in the DNA-specific recognition mechanism by c-Myb besides the AACNG consensuses, which is beneficial to understanding the cell early differentiation and proliferation regulated by c-Myb, as well as the prediction of novel c-Myb-binding motifs in tumorigenesis.

Differences in hepatocellular iron metabolism underlie sexual dimorphism in hepatocyte ferroptosis

Males show higher incidence and severity than females in hepatic injury and many liver diseases, but the mechanisms are not well understood. Ferroptosis, an iron-mediated lipid peroxidation-dependent death, plays an important role in the pathogenesis of liver diseases. We determined whether hepatocyte ferroptosis displays gender difference, accounting for sexual dimorphism in liver diseases. Compared to female hepatocytes, male hepatocytes were much more vulnerable to ferroptosis by iron and pharmacological inducers including RSL3 and iFSP1. Male but not female hepatocytes exhibited significant increases in mitochondrial Fe2+ and mitochondrial ROS (mtROS) contents. Female hepatocytes showed a lower expression of iron importer transferrin receptor 1 (TfR1) and mitochondrial iron importer mitoferrin 1 (Mfrn1), but a higher expression of iron storage protein ferritin heavy chain 1 (FTH1). It is well known that TfR1 expression is positively correlated with ferroptosis. Herein, we showed that silencing FTH1 enhanced while knockdown of Mfrn1 decreased ferroptosis in HepG2 cells. Removing female hormones by ovariectomy (OVX) did not dampen but rather enhanced hepatocyte resistance to ferroptosis. Mechanistically, OVX potentiated the decrease in TfR1 and increase in FTH1 expression. OVX also increased FSP1 expression in ERK-dependent manner. Elevation in FSP1 suppressed mitochondrial Fe2+ accumulation and mtROS production, constituting a novel mechanism of FSP1-mediated inhibition of ferroptosis. In conclusion, differences in hepatocellular iron handling between male and female account, at least in part, for sexual dimorphism in induced ferroptosis of the hepatocytes.

Molecular pathway of mitochondrial preprotein import through the TOM-TIM23 supercomplex.

Over half of mitochondrial proteins are imported from the cytosol via the pre-sequence pathway, controlled by the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. The mechanisms through which proteins are translocated via the TOM and TIM23 complexes remain unclear. Here we report the assembly of the active TOM-TIM23 supercomplex of Saccharomyces cerevisiae with translocating polypeptide substrates. Electron cryo-microscopy analyses reveal that the polypeptide substrates pass the TOM complex through the center of a Tom40 subunit, interacting with a glutamine-rich region. Structural and biochemical analyses show that the TIM23 complex contains a heterotrimer of the subunits Tim23, Tim17 and Mgr2. The polypeptide substrates are shielded from lipids by Mgr2 and Tim17, which creates a translocation pathway characterized by a negatively charged entrance and a central hydrophobic region. These findings reveal an unexpected pre-sequence pathway through the TOM-TIM23 supercomplex spanning the double membranes of mitochondria.

Targeting of CRISPR-Cas12a crRNAs into human mitochondria

Mitochondrial gene editing holds great promise as a therapeutic approach for mitochondrial diseases caused by mutations in the mitochondrial DNA (mtDNA). Current strategies focus on reducing mutant mtDNA heteroplasmy levels through targeted cleavage or base editing. However, the delivery of editing components into mitochondria remains a challenge. Here we investigate the import of CRISPR-Cas12a system guide RNAs (crRNAs) into human mitochondria and study the structural requirements for this process by northern blot analysis of RNA isolated from nucleases-treated mitoplasts. To investigate whether the fusion of crRNA with known RNA import determinants (MLS) improve its mitochondrial targeting, we added MLS hairpin structures at 3′-end of crRNA and demonstrated that this did not impact crRNA ability to program specific cleavage of DNA in lysate of human cells expressing AsCas12a nuclease. Surprisingly, mitochondrial localization of the fused crRNA molecules was not improved compared to non-modified version, indicating that structured scaffold domain of crRNA can probably function as MLS, assuring crRNA mitochondrial import. Then, we designed a series of crRNAs targeting different regions of mtDNA and demonstrated their ability to program specific cleavage of mtDNA fragments in cell lysate and their partial localization in mitochondrial matrix in human cells transfected with these RNA molecules. We hypothesize that mitochondrial import of crRNAs may depend on their secondary structure/sequence. We presume that imported crRNA allow reconstituting the active crRNA/Cas12a system in human mitochondria, which can contribute to the development of effective strategies for mitochondrial gene editing and potential future treatment of mitochondrial diseases.

NLG1, encoding a mitochondrial membrane protein, controls leaf and grain development in rice

BackgroundMitochondrion is the key respiratory organ and participate in multiple anabolism and catabolism pathways in eukaryote. However, the underlying mechanism of how mitochondrial membrane proteins regulate leaf and grain development remains to be further elucidated.ResultsHere, a mitochondria-defective mutant narrow leaf and slender grain 1 (nlg1) was identified from an EMS-treated mutant population, which exhibits narrow leaves and slender grains. Moreover, nlg1 also presents abnormal mitochondria structure and was sensitive to the inhibitors of mitochondrial electron transport chain. Map-based cloning and transgenic functional confirmation revealed that NLG1 encodes a mitochondrial import inner membrane translocase containing a subunit Tim21. GUS staining assay and RT-qPCR suggested that NLG1 was mainly expressed in leaves and panicles. The expression level of respiratory function and auxin response related genes were significantly down-regulated in nlg1, which may be responsible for the declination of ATP production and auxin content.ConclusionsThese results suggested that NLG1 plays an important role in the regulation of leaf and grain size development by maintaining mitochondrial homeostasis. Our finding provides a novel insight into the effects of mitochondria development on leaf and grain morphogenesis in rice.

The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.

Integrative analysis of mitochondrial metabolic reprogramming in early-stage colon and liver cancer.

Gastrointestinal malignancies, including colon adenocarcinoma (COAD) and liver hepatocellular carcinoma (LIHC), remain leading causes of cancer-related deaths worldwide. To better understand the underlying mechanisms of these cancers and identify potential therapeutic targets, we analyzed publicly accessible Cancer Genome Atlas datasets of COAD and LIHC. Our analysis revealed that differentially expressed genes (DEGs) during early tumorigenesis were associated with cell cycle regulation. Additionally, genes related to lipid metabolism were significantly enriched in both COAD and LIHC, suggesting a crucial role for dysregulated lipid metabolism in their development and progression. We also identified a subset of DEGs associated with mitochondrial function and structure, including upregulated genes involved in mitochondrial protein import and respiratory complex assembly. Further, we identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) as a crucial regulator of cancer cell metabolism. Using a genome-scale metabolic model, we demonstrated that HMGCS2 suppression increased glycolysis, lipid biosynthesis, and elongation while decreasing fatty acid oxidation in colon cancer cells. Our study highlights the potential contribution of dysregulated lipid metabolism, including ketogenesis, to COAD and LIHC development and progression and identifies potential therapeutic targets for these malignancies.