Mitochondrial Targeting Sequence Research Articles

Modulation of Antioxidant Enzyme Expression of In Vitro Culture-Derived Reticulocytes

The regulation of reactive oxygen species (ROS) in red blood cells (RBCs) is crucial for maintaining functionality and lifespan. Indeed, dysregulated ROS occurs in haematological diseases such as sickle cell disease and β-thalassaemia. In order to combat this, RBCs possess high levels of protective antioxidant enzymes. We aimed to further boost RBC antioxidant capacity by overexpressing peroxiredoxin (Prxs) and glutathione peroxidase (GPxs) enzymes. Multiple antioxidant enzyme cDNAs were individually overexpressed in expanding immortalised erythroblasts using lentivirus, including Prx isoforms 1, 2, and 6 and GPx isoforms 1 and 4. Enhancing Prx protein expression proved straightforward, but GPx overexpression required modifications. For GPx4, these modifications included adding a SECIS element in the 3’UTR, the removal of a mitochondrial-targeting sequence, and removing putative ubiquitination sites. Culture-derived reticulocytes exhibiting enhanced levels of Prx and GPx antioxidant proteins were successfully engineered, demonstrating a novel approach to improve RBC resilience to oxidative stress. Further work is needed to explore the activity of these proteins and their impact on RBC metabolism, but this strategy shows promise for improving RBC function in physiological and pathological contexts and during storage for transfusion. Enhancing the antioxidant capacity of reticulocytes has exciting promise for developing culture-derived RBCs with enhanced resistance to oxidative damage and offers new therapeutic interventions in diseases with elevated oxidative stress.

Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level.

Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.

Upstream alternative polyadenylation in SCN5A produces a short transcript isoform encoding a mitochondria-localized NaV1.5 N-terminal fragment that influences cardiomyocyte respiration.

SCN5A encodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates action potentials. SCN5A gene variants cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently found a well-conserved alternative polyadenylation (APA) signal downstream of the first SCN5A coding exon. This yields a SCN5A-short transcript isoform expressed in several species (e.g. human, pig, and cat), though rodents lack this upstream APA. Reanalysis of transcriptome-wide cardiac APA-seq and mRNA-seq data shows reductions in both upstream APA usage and short/full-length SCN5A mRNA ratios in failing hearts. Knock-in of the human SCN5A APA sequence into mice is sufficient to enable expression of SCN5A -short transcript, while significantly decreasing expression of full-length SCN5A mRNA. Notably, SCN5A -short transcript encodes a novel protein (NaV1.5-NT), composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from intronic sequence. AAV9 constructs were able to achieve stable NaV1.5-NT expression in mouse hearts, and western blot of human heart tissues showed bands co-migrating with NaV1.5-NT transgene-derived bands. NaV1.5-NT is predicted to contain a mitochondrial targeting sequence and localizes to mitochondria in cultured cardiomyocytes and in mouse hearts. NaV1.5-NT expression in cardiomyocytes led to elevations in basal oxygen consumption rate, ATP production, and mitochondrial ROS, while depleting NADH supply. Native PAGE analyses of mitochondria lysates revealed that NaV1.5-NT expression resulted in increased levels of disassembled complex V subunits and accumulation of complex I-containing supercomplexes. Overall, we discovered that APA-mediated regulation of SCN5A produces a short transcript encoding NaV1.5-NT. Our data support that NaV1.5-NT plays a multifaceted role in influencing mitochondrial physiology: 1) by increasing basal respiration likely through promoting complex V conformations that enhance proton leak, and 2) by increasing overall respiratory efficiency and NADH consumption by enhancing formation and/or stability of complex I-containing respiratory supercomplexes, though the specific molecular mechanisms underlying each of these remain unresolved.

Mitochondrial N‐Terminal Signal Sequences as Novel Antimicrobial and Annticancer Peptides

AbstractN‐terminal mitochondrial targeting sequences (MTS) are signal peptides to drive the localization and import of mitochondrial protein and exhibit an amphipathic α‐helix structure similar to that of almost all antimicrobial peptides (AMPs). Here, 2,939 mitochondrial proteins are excavated from various organisms, and 574 MTS are selected with an MTS possibility score exceeding 0.5, as determined via MitoFates (http://mitf.cbrc.jp/MitoFates/cgi‐bin/top.cgi). 26 MTS composed of less than 20 amino acids is synthesized and analyzed their antimicrobial activities. The results showed that approximately 50% of MTS exhibited antimicrobial activity. Among them, human mitochondrial transcription termination factor 2 (hMTERF2‐ts) is identified as an AMP candidate with strong antimicrobial activity against gram‐positive and gram‐negative bacteria, antibiotics‐resistant (AR) bacteria, and fungi. Furthermore, it shows anticancer activities against various cancer cells, such as MDA‐MB‐231, HT‐29, HepG2, and Panc‐1 cells, with high stability against proteases, heat, pH, and salt, and low cytotoxicity toward normal cells, HaCaT and RAW 264.7. In addition, hMTERF2‐ts effectively prevented the growth of Panc‐1‐implanted tumors without weight loss in BALB/c nude mice. In conclusion, this study suggests that MTSs of protozoa are powerful and novel AMP candidates with low cytotoxicity against normal cells.

Upgrading Mitochondria-Targeting Peptide-Based Nanocomplexes for Zebrafish In Vivo Compatibility Assays.

The lack of effective delivery systems has slowed the development of mitochondrial gene therapy. Delivery systems based on cell-penetrating peptides (CPPs) like the WRAP (tryptophan and arginine-rich peptide) family conjugated with a mitochondrial targeting sequence (MTS) have emerged as adequate carriers to mediate gene expression into the mitochondria. In this work, we performed the PEGylation of WRAP/pDNA nanocomplexes and compared them with previously analyzed nanocomplexes such as (KH)9/pDNA and CpMTP/pDNA. All nanocomplexes exhibited nearly homogeneous sizes between 100 and 350 nm in different environments. The developed complexes were biocompatible and hemocompatible to both human astrocytes and lung smooth muscle cells, ensuring in vivo safety. The nanocomplexes displayed mitochondria targeting ability, as through transfection they preferentially accumulate into the mitochondria of astrocytes and muscle cells to the detriment of cytosol and lysosomes. Moreover, the transfection of these cells with MTS-CPP/pDNA complexes produced significant levels of mitochondrial protein ND1, highlighting their efficient role as gene delivery carriers toward mitochondria. The positive obtained data pave the way for in vivo research. Using confocal microscopy, the cellular internalization capacity of these nanocomplexes in the zebrafish embryo model was assessed. The peptide-based nanocomplexes were easily internalized into zebrafish embryos, do not cause harmful or toxic effects, and do not affect zebrafish's normal development and growth. These promising results indicate that MTS-CPP complexes are stable nanosystems capable of internalizing in vivo models and do not present associated toxicity. This work, even at an early stage, offers good prospects for continued in vivo zebrafish research to evaluate the performance of nanocomplexes for mitochondrial gene therapy.

The cytosolic form of dual localized BolA family protein Bol3 is important for adaptation to iron starvation in Aspergillus fumigatus.

Aspergillus fumigatus is the predominant mould pathogen for humans. Adaption to host-imposed iron limitation has previously been demonstrated to be essential for its virulence. [2Fe-2S] clusters are crucial as cofactors of several metabolic pathways and mediate cytosolic/nuclear iron sensing in fungi including A. fumigatus. [2Fe-2S] cluster trafficking has been shown to involve BolA family proteins in both mitochondria and the cytosol/nucleus. Interestingly, both A. fumigatus homologues, termed Bol1 and Bol3, possess mitochondrial targeting sequences, suggesting the lack of cytosolic/nuclear versions. Here, we show by the combination of mutational, proteomic and fluorescence microscopic analyses that expression of the Bol3 encoding gene leads to dual localization of gene products to mitochondria and the cytosol/nucleus via alternative translation initiation downstream of the mitochondrial targeting sequence, which appears to be highly conserved in various Aspergillus species. Lack of either mitochondrial Bol1 or Bol3 was phenotypically inconspicuous while lack of cytosolic/nuclear Bol3 impaired growth during iron limitation but not iron sensing which indicates a particular importance of [2Fe-2S] cluster trafficking during iron limitation. Remarkably, cytosolic/nuclear Bol3 differs from the mitochondrial version only by N-terminal acetylation, a finding that was only possible by mutational hypothesis testing.

Evolutionary Changes in Primate Glutamate Dehydrogenases 1 and 2 Influence the Protein Regulation by Ligands, Targeting and Posttranslational Modifications.

There are two paralogs of glutamate dehydrogenase (GDH) in humans encoded by the GLUD1 and GLUD2 genes as a result of a recent retroposition during the evolution of primates. The two human GDHs possess significantly different regulation by allosteric ligands, which is not fully characterized at the structural level. Recent advances in identification of the GDH ligand binding sites provide a deeper perspective on the significance of the accumulated substitutions within the two GDH paralogs. In this review, we describe the evolution of GLUD1 and GLUD2 after the duplication event in primates using the accumulated sequencing and structural data. A new gibbon GLUD2 sequence questions the indispensability of ancestral R496S and G509A mutations for GLUD2 irresponsiveness to GTP, providing an alternative with potentially similar regulatory features. The data of both GLUD1 and GLUD2 evolution not only confirm substitutions enhancing GLUD2 mitochondrial targeting, but also reveal a conserved mutation in ape GLUD1 mitochondrial targeting sequence that likely reduces its transport to mitochondria. Moreover, the information of GDH interactors, posttranslational modification and subcellular localization are provided for better understanding of the GDH mutations. Medically significant point mutations causing deregulation of GDH are considered from the structural and regulatory point of view.

Dual cytoplasmic-mitochondrial engineering for improved patchoulol production in Komagataella phaffii

Mitochondria are a potential sub-organelle for the synthesis of terpenoids due to its abundant acetyl-CoA, cofactors, and compact compartment for targeting metabolic pathways and enzymes. Hence, conducting systematic investigations and harnessing the subcellular compartment of mitochondria may offer a viable avenue for enhancing terpenoids biosynthesis in yeast and other eukaryotic cells. Previous endeavors to boost terpenoids production in Komagataella phaffii mainly focused on diverse metabolic engineering strategies in the cytoplasm. Numerous studies have demonstrated that reconstructing specific metabolic pathways within mitochondria can lead to higher production yields than engineering the same pathways in the cytoplasm. In our study, we initially identified 6 well-performed mitochondrial targeting sequences (MTSs) through colocalization observation of EGFP and a mitochondrial red fluorescent probe via fluorescence microscopy. These MTSs were selected for the assembly of complete patchoulol biosynthesis pathways in the mitochondria, which resulted in a 1.7-fold increase in patchoulol production compared to the original strain. Furthermore, dual regulation of the cytoplasm and mitochondria was implemented in the P6H4＋PM2 strain to further enhance patchoulol synthesis to 109 mg/L, representing a 1.0-fold and 10.7-fold greater yield than that of the strains engineered solely in the cytoplasm or mitochondria, respectively. This study offers valuable guidance for effectively utilizing mitochondria to achieve higher patchoulol and other sesquiterpenes through dual cytoplasmic-mitochondrial engineering in K. phaffii.

Identification of a novel MT-ND3 variant and restoring mitochondrial function by allotopic expression of MT-ND3 gene

Mitochondrial diseases are caused by nuclear, or mitochondrial DNA (mtDNA) variants and related co-factors. Here, we report a novel m.10197G > C variant in MT-ND3 in a patient, and two other patients with m.10191 T > C. MT-ND3 variants are known to cause Leigh syndrome or mitochondrial complex I deficiency. We performed the functional analyses of the novel m.10197G > C variant that significantly lowered MT-ND3 protein levels, causing complex I assembly and activity deficiency, and reduction of ATP synthesis. We adapted a previously described re-engineering technique of delivering mitochondrial genes into mitochondria through codon optimization for nuclear expression and translation by cytoplasmic ribosomes to rescue defects arising from the MT-ND3 variants. We constructed mitochondrial targeting sequences along with the codon-optimized MT-ND3 and imported them into the mitochondria. To achieve the goal, we imported codon-optimized MT-ND3 into mitochondria in three patients with m.10197G > C and m.10191 T > C missense variants in the MT-ND3. Nuclear expression of the MT-ND3 gene partially restored protein levels, complex I deficiency, and significant improvement of ATP production indicating a functional rescue of the mutant phenotype. The codon-optimized nuclear expression of mitochondrial protein and import inside the mitochondria can supplement the requirements for ATP in energy-deficient mitochondrial disease patients.

Salmonella enterica serovar Typhimurium remodels mitochondrial dynamics of macrophages via the T3SS effector SipA to promote intracellular proliferation

ABSTRACT Mitochondrial dynamics are critical in cellular energy production, metabolism, apoptosis, and immune responses. Pathogenic bacteria have evolved sophisticated mechanisms to manipulate host cells’ mitochondrial functions, facilitating their proliferation and dissemination. Salmonella enterica serovar Typhimurium (S. Tm), an intracellular foodborne pathogen, causes diarrhea and exploits host macrophages for survival and replication. However, S. Tm-associated mitochondrial dynamics during macrophage infection remain poorly understood. In this study, we showed that within macrophages, S. Tm remodeled mitochondrial fragmentation to facilitate intracellular proliferation mediated by Salmonella invasion protein A (SipA), a type III secretion system effector encoded by Salmonella pathogenicity island 1. SipA directly targeted mitochondria via its N-terminal mitochondrial targeting sequence, preventing excessive fragmentation and the associated increase in mitochondrial reactive oxygen species, loss of mitochondrial membrane potential, and release of mitochondrial DNA and cytochrome c into the cytosol. Macrophage replication assays and animal experiments showed that mitochondria and SipA interact to facilitate intracellular replication and pathogenicity of S. Tm. Furthermore, we showed that SipA delayed mitochondrial fragmentation by indirectly inhibiting the recruitment of cytosolic dynamin-related protein 1, which mediates mitochondrial fragmentation. This study revealed a novel mechanism through which S. Tm manipulates host mitochondrial dynamics, providing insights into the molecular interplay that facilitates S. Tm adaptation within host macrophages.

Mitochondrial HER2 stimulates respiration and promotes tumorigenicity.

Amplification of HER2, a receptor tyrosine kinase and a breast cancer-linked oncogene, is associated with aggressive disease. HER2 protein is localised mostly at the cell membrane, but a fraction translocates to mitochondria. Whether and how mitochondrial HER2 contributes to tumorigenicity is currently unknown. We enriched the mitochondrial (mt-)HER2 fraction in breast cancer cells using an N-terminal mitochondrial targeting sequence and analysed how this manipulation impacts bioenergetics and tumorigenic properties. The role of the tyrosine kinase activity of mt-HER2 was assessed in wild type, kinase-dead (K753M) and kinase-enhanced (V659E) mtHER2 constructs. We document that mt-HER2 associates with the oxidative phosphorylation system, stimulates bioenergetics and promotes larger respiratory supercomplexes. mt-HER2 enhances proliferation and invasiveness invitro and tumour growth and metastatic potential invivo, in a kinase activity-dependent manner. On the other hand, constitutively active mt-HER2 provokes excessive mitochondria ROS generation, sensitises to cell death, and restricts growth of primary tumours, suggesting that regulation of HER2 activity in mitochondria is required for the maximal pro-tumorigenic effect. mt-HER2 promotes tumorigenicity by supporting bioenergetics and optimal redox balance.

Sua5 catalyzing universal t6A tRNA modification is responsible for multifaceted functions of the KEOPS complex in Cryptococcus neoformans.

The N6-threonylcarbamoyl adenosine (t6A) tRNA modification is critical for ensuring translation fidelity across three domains of life. Our prior work highlighted the KEOPS complex, organized in a Pcc1-Kae1-Bud32-Cgi121 linear arrangement, not only serves an evolutionarily conserved role in t6A tRNA modification but also exerts diverse functional impacts on pathobiological characteristics in Cryptococcus neoformans, a leading cause of fungal meningitis worldwide. However, the extent to which the pleiotropic functions of the KEOPS complex are specifically tied to tRNA modification remains uncertain. To address this, we undertook a functional characterization of Sua5, responsible for generating the precursor threonylcarbamoyl-adenylate (TC-AMP) for t6A tRNA modification, using a reverse genetics approach. Comparative phenotypic analyses with KEOPS mutants revealed that Sua5 plays a vital role in multiple cellular processes, such as t6A tRNA modification, growth, sexual development, stress response, and virulence factor production, thus reflecting the multifaceted functions of the KEOPS complex. In support of this, sua5Δ bud32Δ double mutants showed phenotypes comparable to those of the corresponding single mutants. Intriguingly, a SUA5 allele lacking a mitochondria targeting sequence (SUA5MTSΔ) was sufficient to restore the wild-type phenotypes in the sua5Δ mutant, suggesting that Sua5's primary functional locus may be cytosolic, akin to the KEOPS complex. Further supporting this, the deletion of Qri7, a mitochondrial paralog of Kae1, had no discernible phenotypic impact on C. neoformans. We concluded that cytosolic t6A tRNA modifications, orchestrated by Sua5 and the KEOPS complex, are central to the regulation of diverse pathobiological functions in C. neoformans.IMPORTANCEUnderstanding cellular functions at the molecular level is crucial for advancing disease treatments. Our research reveals a critical connection between the KEOPS complex and Sua5 in Cryptococcus neoformans, a significant cause of fungal meningitis. While the KEOPS complex is known for its versatile roles in cellular processes, Sua5 is specialized in t6A tRNA modification. Our key finding is that the diverse roles of the KEOPS complex, ranging from cell growth and stress response to virulence, are fundamentally linked to its function in t6A tRNA modification. This conclusion is supported by the remarkable similarities between the impacts of Sua5 and KEOPS on these processes, despite their roles in different steps of the t6A modification pathway. This newfound understanding deepens our insight into fungal biology and opens new avenues for developing potential therapies against dangerous fungal diseases.

Mitochondrial DNA editing in potato through mitoTALEN and mitoTALECD: molecular characterization and stability of editing events

BackgroundThe aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125).ResultsTargeted chondriome deletions of different sizes (236–1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11–12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring.ConclusionsBoth editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.

2',3' cyclic nucleotide 3' phosphodiesterase 1 functional isoform antagonizes HIV-1 particle assembly.

IFN-stimulated gene 2',3' cyclic nucleotide 3' phosphodiesterase (CNP) comprises two isoforms: the short CNP1 and the long CNP2, featuring an additional N-terminal segment of 20 amino acids (N20aa) proposed as a mitochondrial targeting sequence. Notably, CNP1 can be produced by cleaving the N20aa segment from CNP2. Although previous investigations have recognized the HIV-1 particle assembly impairment capability of CNP2, the antiviral activity of CNP1 remains ambiguous. Our study clarifies that CNP1, as opposed to CNP2, serves as the primary isoform exerting anti-HIV-1 activity. Both CNP1 and CNP2 can localize to the cell membrane, but the N20aa segment of CNP2 impedes CNP2-HIV-1 Gag interaction. Cleavage of the N20aa segment from CNP2 results in the formation of a functional, truncated form known as CNP1. Intriguingly, this posttranslational processing of CNP2 N20aa occurs within the cytoplasmic matrix rather than the mitochondria. Regulated by CTII motif prenylation, CNP1 proteins translocate to the cell membrane and engage with HIV-1 Gag. In conclusion, our findings underscore the pivotal role of posttranslational modification in governing the inhibitory potential of CNP in HIV-1 replication.

Methods for Monitoring Mitophagy Using mt-Keima.

Mitochondria-targeted Keima (mt-Keima) is a pH-sensitive, acid-stable fluorescent protein used for the quantification of mitophagy. Mt-Keima contains a mitochondrial matrix targeting sequence and has bimodal excitation with peaks at 440nM in neutral environments and 586nM in acidic environments. From this bimodal excitation, a ratiometric signal may be calculated to quantify mitophagy in live cells. This chapter describes procedures for measuring mitophagy by flow cytometry and live cell confocal microscopy with mt-Keima.

Mitochondrial targeting sequence of magnetoreceptor MagR: More than just targeting.

Iron-sulfur clusters are essential cofactors for proteins involved in various biological processes, such as electron transport, biosynthetic reactions, DNA repair, and gene expression regulation. Iron-sulfur cluster assembly protein IscA1 (or MagR) is found within the mitochondria of most eukaryotes. Magnetoreceptor (MagR) is a highly conserved A-type iron and iron-sulfur cluster-binding protein, characterized by two distinct types of iron-sulfur clusters, [2Fe-2S] and [3Fe-4S], each conferring unique magnetic properties. MagR forms a rod-like polymer structure in complex with photoreceptive cryptochrome (Cry) and serves as a putative magnetoreceptor for retrieving geomagnetic information in animal navigation. Although the N-terminal sequences of MagR vary among species, their specific function remains unknown. In the present study, we found that the N-terminal sequences of pigeon MagR, previously thought to serve as a mitochondrial targeting signal (MTS), were not cleaved following mitochondrial entry but instead modulated the efficiency with which iron-sulfur clusters and irons are bound. Moreover, the N-terminal region of MagR was required for the formation of a stable MagR/Cry complex. Thus, the N-terminal sequences in pigeon MagR fulfil more important functional roles than just mitochondrial targeting. These results further extend our understanding of the function of MagR and provide new insights into the origin of magnetoreception from an evolutionary perspective.

A mitophagy sensor PPTC7 controls BNIP3 and NIX degradation to regulate mitochondrial mass

Mitophagy mediated by BNIP3 and NIX critically regulates mitochondrial mass. Cellular BNIP3 and NIX levels are tightly controlled by SCFFBXL4-mediated ubiquitination to prevent excessive mitochondrial loss and lethal disease. Here, we report that knockout of PPTC7, a mitochondrial matrix protein, hyperactivates BNIP3-/NIX-mediated mitophagy and causes perinatal lethality that is rescued by NIX knockout in mice. Biochemically, the PPTC7 precursor is trapped by BNIP3 and NIX to the mitochondrial outer membrane, where PPTC7 scaffolds assembly of a substrate-PPTC7-SCFFBXL4 holocomplex to degrade BNIP3 and NIX, forming a homeostatic regulatory loop. PPTC7 possesses an unusually weak mitochondrial targeting sequence to facilitate its outer membrane retention and mitophagy control. Starvation upregulates PPPTC7 expression in mouse liver to repress mitophagy, which critically maintains hepatic mitochondrial mass, bioenergetics, and gluconeogenesis. Collectively, PPTC7 functions as a mitophagy sensor that integrates homeostatic and physiological signals to dynamically control BNIP3 and NIX degradation, thereby maintaining mitochondrial mass and cellular homeostasis.

Generation of inducible mitophagy mice.

Mitophagy is programmed selective autophagy of mitochondria and is important for mitochondrial quality control and cellular homeostasis. Mitochondrial dysfunction and impaired mitophagy are closely associated with various diseases, including heart failure and diabetes. To better understand the pathophysiological role of mitophagy, we generated doxycycline-inducible mitophagy mice using a synthetic mitophagy adaptor protein consisting of an outer mitochondrial membrane targeting sequence and an engineered LIR. To evaluate the activation of mitophagy upon doxycycline treatment, we also generated mitophagy reporter mito-QC mice in which mitochondria tandemly express mCherry and GFP, and only GFP signals are lost in acidic lysosomes subjected to mitophagy. With the ROSA26 promoter-driven rtTA, mitophagy was observed at least in heart, liver, and skeletal muscle. We investigated the relationship between mitophagy activation and pressure overload heart failure or high fat diet-induced obesity. Unexpectedly, we were unable to confirm the protective effect of mitophagy in these two pathological models. Further titration of the level of mitophagy induction is required to demonstrate the potency of the protective effects of mitophagy in disease models.

Degradation of citrate synthase lacking the mitochondrial targeting sequence is inhibited in cells defective in Hsp70/Hsp40 chaperones under heat stress conditions.

Most nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N∆Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCFUcc1 ubiquitin ligase complex. Here, we show that Hsp70 and Hsp40 chaperones (Ssa1 and Ydj1 in yeast, respectively) are required for N∆Cit1 degradation under heat stress conditions. In the absence of Hsp70 function, a portion of N∆Cit1-GFP formed insoluble aggregates and cytosolic foci. However, the extent of ubiquitination of N∆Cit1 was unaffected, implying that Hsp70/Hsp40 chaperones are involved in the postubiquitination step of N∆Cit1 degradation. Intriguingly, degradation of cytosolic/peroxisomal gluconeogenic citrate synthase (Cit2), an endogenous substrate for SCFUcc1-mediated proteasomal degradation, was not highly dependent on Hsp70 even under heat stress conditions. These results suggest that mitochondrial citrate synthase is thermally vulnerable in the cytosol, where Hsp70/Hsp40 chaperones are required to facilitate its degradation.

Perilipin 5 protects the mitochondrial oxidative functions and improves the alcoholic liver injury in mice.

Alcohol consumption is a well-established risk factor for the onset and progression of hepatic steatosis. Perilipin 5 (Plin5), a lipid droplet protein, is an important protective factor against hepatic lipotoxicity induced by excessive lipolysis, but its role and molecular mechanism in alcoholic liver disease (ALD) are not fully elucidated. The optimized National Institute on Alcohol Abuse and Alcoholism model was used to construct ALD model mice. Automatic biochemical analyser was used for Biochemical Parameters. The primary hepatocytes and Plin5-overexpressed HepG2 cells (including full-length Plin5 and Plin5 deleting 444-464 aa) were used for invitro experiment. Haematoxylin and Eosin staining, Oil Red O staining, Bodipy 493/503 staining, Periodic Acid-Schiff staining, immunohistochemistry and JC-1 staining were used to evaluate cell morphology, lipids, glycogen, inflammation and membrane potential. Commercially kits are used to detect glycolipid metabolites, such as triglycerides, glycogen, glucose, reactive oxygen species, lactic acids, ketone bodies. Fluorescently labelled deoxyglucose, NBDG, was used for glucose intake. An XF96 extracellular flux analyser was used to determinate oxygen consumption rate in hepatocytes. The morphological and structural damage of mitochondria was evaluated by electron microscopy. Classical ultracentrifugation is used to separate the subcellular organelles of tissues and cells. Immunoblotting and qPCR were used to detect changes in mRNA and protein levels of related genes. Our results showed that the expression of Plin5 in mouse livers was enhanced by alcohol intake, and Plin5 deficiency aggravated the alcohol-induced liver injury. To clarify the mechanism, we found that Plin5 deficiency significantly elevated the hepatic NADH levels and ketone body production in the alcohol-treated mice. As NADH elevation could promote the reduction of pyruvate into lactate and then inhibit the gluconeogenesis, alcohol-treated Plin5-deficient mice exhibited more lactate production and severer hypoglycemia. These results implied that Plin5 deficiency impaired the mitochondrial oxidative functions in the presence of alcohol. In addition, we demonstrated that Plin5 could be recruited onto mitochondria by alcohol, while Plin5 without mitochondrial targeting sequences lost its mitochondrial protection functions. Collectively, this study demonstrated that the mitochondrial Plin5 could protect the alcohol-induced mitochondrial injury, which provides an important new insight on the roles of Plin5 in highly oxidative tissues.