Mitochondrial Localization Signal Research Articles

Dual-localization signals enhance mitochondrial targeted presentation of engineered proteins.

Effective delivery of engineered proteins into mitochondria is of great significance for developing efficient mitochondrial DNA editing tools and realizing accurate treatment of mitochondrial diseases. Here, the candidate genes, eGFP and Cas9, were engineered with different mitochondrial localization signal (MLS) sequences introduced at their up- or/and down-streams. The corresponding expression vectors for the engineered proteins were constructed respectively, and HEK293T cells were transfected with these vectors. The fluorescence colocalization and Western blotting assays were used to analyze the mitochondrial targeting presentation effect of different engineered proteins. The results demonstrated that the daul-MLS modification of the eGFP and Cas9 proteins significantly improved the efficiency of mitochondrial targeted presentation, compared with the engineered proteins with single MLS added. Hence, it is speculated that dual MLS strategy can enhance the mitochondrial targeting of engineered proteins, which lays a theoretical foundation for the future development of efficient mitochondrial DNA editing tools.

A Physiological Approach to Explore How Thioredoxin-Glutathione Reductase (TGR) and Peroxiredoxin (Prx) Eliminate H2O2 in Cysticerci of Taenia.

Peroxiredoxins (Prxs) and glutathione peroxidases (GPxs) are the main enzymes of the thiol-dependent antioxidant systems responsible for reducing the H2O2 produced via aerobic metabolism or parasitic organisms by the host organism. These antioxidant systems maintain a proper redox state in cells. The cysticerci of Taenia crassiceps tolerate millimolar concentrations of this oxidant. To understand the role played by Prxs in this cestode, two genes for Prxs, identified in the genome of Taenia solium (TsPrx1 and TsPrx3), were cloned. The sequence of the proteins suggests that both isoforms belong to the class of typical Prxs 2-Cys. In addition, TsPrx3 harbors a mitochondrial localization signal peptide and two motifs (-GGLG- and -YP-) associated with overoxidation. Our kinetic characterization assigns them as thioredoxin peroxidases (TPxs). While TsPrx1 and TsPrx3 exhibit the same catalytic efficiency, thioredoxin-glutathione reductase from T. crassiceps (TcTGR) was five and eight times higher. Additionally, the latter demonstrated a lower affinity (>30-fold) for H2O2 in comparison with TsPrx1 and TsPrx3. The TcTGR contains a Sec residue in its C-terminal, which confers additional peroxidase activity. The aforementioned aspect implies that TsPrx1 and TsPrx3 are catalytically active at low H2O2 concentrations, and the TcTGR acts at high H2O2 concentrations. These results may explain why the T. crassiceps cysticerci can tolerate high H2O2 concentrations.

Rapid and accurate identification of stem cell differentiation stages via SERS and convolutional neural networks

Monitoring the transition of cell states during induced pluripotent stem cell (iPSC) differentiation is crucial for clinical medicine and basic research. However, both identification category and prediction accuracy need further improvement. Here, we propose a method combining surface-enhanced Raman spectroscopy (SERS) with convolutional neural networks (CNN) to precisely identify and distinguish cell states during stem cell differentiation. First, mitochondria-targeted probes were synthesized by combining AuNRs and mitochondrial localization signal (MLS) peptides to obtain effective and stable SERS spectra signals at various stages of cell differentiation. Then, the SERS spectra served as input datasets, and their distinctive features were learned and distinguished by CNN. As a result, rapid and accurate identification of six different cell states, including the embryoid body (EB) stage, was successfully achieved throughout the stem cell differentiation process with an impressive prediction accuracy of 98.5%. Furthermore, the impact of different spectral feature peaks on the identification results was investigated, which provides a valuable reference for selecting appropriate spectral bands to identify cell states. This is also beneficial for shortening the spectral acquisition region to enhance spectral acquisition speed. These results suggest the potential for SERS-CNN models in quality monitoring of stem cells, advancing the practical applications of stem cells.

Cholinergic Receptor Muscarinic 1 Co-Localized with Mitochondria in Cultured Dorsal Root Ganglion Neurons, and Its Deletion Disrupted Mitochondrial Ultrastructure in Peripheral Neurons: Implications in Alzheimer's Disease.

Loss of Cholinergic Receptor Muscarinic 1 (CHRM1) has been linked to the pathogenesis of Alzheimer's disease (AD). Our recent study found significantly lower CHRM1 protein levels in AD patient cortices, linked to reduced survival. Furthermore, using knockout mice (Chrm1-/-) we demonstrated that deletion of Chrm1 alters cortical mitochondrial structure and function, directly establishing a connection between its loss and mitochondrial dysfunction in the context of AD. While CHRM1's role in the brain has been extensively investigated, its impact on peripheral neurons in AD remains a crucial area of research, especially considering reported declines in peripheral nerve conduction among AD patients. The objective was to characterize Chrm1 localization and mitochondrial deficits in Chrm1-/- dorsal root ganglion (DRG) neurons. Recombinant proteins tagged with Green or Red Fluorescent Protein (GFP/RFP) were transiently expressed to investigate the localization of Chrm1 and mitochondria, as well as mitochondrial movement in the neurites of cultured primary mouse DRG neurons, using confocal time-lapse live cell imaging. Transmission electron microscopy was performed to examine the ultrastructure of mitochondria in both wild-type and Chrm1-/- DRGs. Fluorescence imaging revealed colocalization and comigration of N-terminal GFP-tagged Chrm1 and mitochondrial localization signal peptide-tagged RFP-labelled mitochondria in the DRGs neurons. A spectrum of mitochondrial structural abnormalities, including disruption and loss of cristae was observed in 87% neurons in Chrm1-/- DRGs. This study suggests that Chrm1 may be localized in the neuronal mitochondria and loss of Chrm1 in peripheral neurons causes sever mitochondrial structural aberrations resembling AD pathology.

AKT1 phosphorylation of cytoplasmic ME2 induces a metabolic switch to glycolysis for tumorigenesis

Many types of tumors feature aerobic glycolysis for meeting their increased energetic and biosynthetic demands. However, it remains still unclear how this glycolytic phenomenon is achieved and coordinated with other metabolic pathways in tumor cells in response to growth stimuli. Here we report that activation of AKT1 induces a metabolic switch to glycolysis from the mitochondrial metabolism via phosphorylation of cytoplasmic malic enzyme 2 (ME2), named ME2fl (fl means full length), favoring an enhanced glycolytic phenotype. Mechanistically, in the cytoplasm, AKT1 phosphorylates ME2fl at serine 9 in the mitochondrial localization signal peptide at the N-terminus, preventing its mitochondrial translocation. Unlike mitochondrial ME2, which accounts for adjusting the tricarboxylic acid (TCA) cycle, ME2fl functions as a scaffold that brings together the key glycolytic enzymes phosphofructokinase (PFKL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2), as well as Lactate dehydrogenase A (LDHA), to promote glycolysis in the cytosol. Thus, through phosphorylation of ME2fl, AKT1 enhances the glycolytic capacity of tumor cells in vitro and in vivo, revealing an unexpected role for subcellular translocation switching of ME2 mediated by AKT1 in the metabolic adaptation of tumor cells to growth stimuli.

Induce male sterility by CRISPR/Cas9-mediated mitochondrial genome editing in tobacco.

Genome editing has become more and more popular in animal and plant systems following the emergence of CRISPR/Cas9 technology. However, target sequence modification by CRISPR/Cas9 has not been reported in the plant mitochondrial genome, mtDNA. In plants, a type of male sterility known as cytoplasmic male sterility (CMS) has been associated with certain mitochondrial genes, but few genes have been confirmed by direct mitochondrial gene-targeted modifications. Here, the CMS-associated gene (mtatp9) in tobacco was cleaved using mitoCRISPR/Cas9 with a mitochondrial localization signal. The male-sterile mutant, with aborted stamens, exhibited only 70% of the mtDNA copy number of the wild type and exhibited an altered percentage of heteroplasmic mtatp9 alleles; otherwise, the seed setting rate of the mutant flowers was zero. Transcriptomic analyses showed that glycolysis, tricarboxylic acid cycle metabolism and the oxidative phosphorylation pathway, which are all related to aerobic respiration, were inhibited in stamens of the male-sterile gene-edited mutant. In addition, overexpression of the synonymous mutations dsmtatp9 could restore fertility to the male-sterile mutant. Our results strongly suggest that mutation of mtatp9 causes CMS and that mitoCRISPR/Cas9 can be used to modify the mitochondrial genome of plants.

Norovirus MLKL-like protein initiates cell deathto induce viral egress.

Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but there is no known mechanism that causes norovirus infection-triggered cell death and lysis1-3. Here we identify a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase NS3 contains an N-terminal four-helix bundle domain homologous to the membrane-disruption domain of the pseudokinase mixed lineage kinase domain-like (MLKL). NS3 has a mitochondrial localization signal and thus induces cell death by targeting mitochondria. Full-length NS3 and an N-terminal fragment of the protein bound the mitochondrial membrane lipid cardiolipin, permeabilized the mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NS3 were essential for cell death, viral egress from cells and viral replication in mice. These findings suggest that noroviruses have acquired a host MLKL-like pore-forming domain to facilitate viral egress by inducing mitochondrial dysfunction.

Methyltransferase METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs

A subset of eukaryotic tRNAs is methylated in the anticodon loop, forming 3-methylcytosine (m3C) modifications. In mammals, the number of tRNAs containing m3C modifications has been expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. However, whereas the enzymes catalyzing m3C formation in nuclear-encoded tRNAs have been identified, the proteins responsible for m3C modification in mt-tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase, as well as with mt-tRNAs containing m3C. We demonstrate that human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs, but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells could be rescued by re-expression of WT METTL8, but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, we found METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA, suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mt-tRNAs and uncover a potential function for m3C modification in mitochondrial tRNA structure.

Subcellular fractionation of brain tumor stem cells.

Brain tumor stem cells (BTSCs) are a rare population of self-renewing stem cells that are cultured as spheres and are often slow growing compared to other mammalian cell lines. Analysis of BTSC proteome requires careful handling as well as techniques that can be applied to small quantities of cell material. Subcellular fractionation is a widely used technique to assess protein localization. Although proteins are often destined to a defined cell compartment via a signal peptide such as mitochondrial or nuclear localization signals, the recruitment of a protein from one compartment to another can occur as a result of post-translational modification and/or structural variations in response to intracellular and extracellular stimuli. These events assign different functions to a protein making the study of protein localization a useful approach for better understanding of its role in disease progression. Sequential centrifugation remains a simple and versatile fractionation method for proteomic analysis. It can also be applied for diverse downstream applications such as multi-omics using pure nuclear fractions or metabolomic studies on isolated mitochondria. In this chapter, we describe our optimized protocol for subcellular fractionation of BTSC spheres in which we use a commercially available kit with additional centrifugation steps. We provide details on BTSC maintenance and handling, fractionation protocol and evaluation of fraction purity.

Identification of uncharacterized proteins potentially localized to mitochondria (UPMs) in Saccharomyces cerevisiae using a fluorescent protein unstable in the cytoplasm.

Eukaryotic cells are composed of organelles, and each organelle contains proteins that play a role in its function. Therefore, the localization of a protein, especially to organelles, is a clue to infer the function of that protein. In this study, we attempted to identify novel mitochondrially localized proteins in the budding yeast Saccharomyces cerevisiae using a fluorescent protein (GFPdeg) that is rapidly degraded in the cytoplasm. Of the budding yeast proteins predicted to localize to mitochondria by the prediction tool Deeploc-1.0, those with known mitochondrial localization or functional relevance were eliminated, and 95 proteins of unknown function were selected as candidates for analysis. By forced expression of GFPdeg fusion proteins with these proteins and observation of their localization, we identified 35 uncharacterized proteins potentially localized to mitochondria (UPMs) including 8 previously identified proteins that localize to mitochondria. Most of these had no N-terminal mitochondrial localization signal and were evolutionarily young "emerging genes" that exist only in S. cerevisiae. Some of these genes were found to be upregulated during the postdiauxic shift phase when mitochondria are being developed, suggesting that they are actually involved in some mitochondrial function.

Mitochondrial Localization Signal of Porcine Circovirus Type 2 Capsid Protein Plays a Critical Role in Cap-Induced Apoptosis.

Porcine circovirus 2 (PCV2), considered one of the most globally important porcine pathogens, causes postweaning multisystemic wasting syndrome (PMWS). This virus is localized in the mitochondria in pigs with PMWS. Here, we identified, for the first time, a mitochondrial localization signal (MLS) in the PCV2 capsid protein (Cap) at the N-terminus. PK-15 cells showed colocalization of the MLS-EGFP fusion protein with mitochondria. Since the PCV2 Cap also contained a nuclear localization signal (NLS) that mediated entry into the nucleus, we inferred that the subcellular localization of the PCV2 Cap is inherently complex and dependent on the viral life cycle. Furthermore, we also determined that deletion of the MLS attenuated Cap-induced apoptosis. More importantly, the MLS was essential for PCV2 replication, as absence of the MLS resulted in failure of virus rescue from cells infected with infectious clone DNA. In conclusion, the MLS of the PCV2 Cap plays critical roles in Cap-induced apoptosis, and MLS deletion of Cap is lethal for virus rescue.

MitoTALENs: A Method for Targeted Gene Disruption in Plant Mitochondrial Genomes.

Modification of plant mitochondrial genomes is still a difficult task, especially in multicellular plants. Transcription activator-like effector nucleases with a mitochondrial localization signal (mitoTALENs) can cut out a desired sequence from the mitochondrial genome in plants. Although vector construction of mitoTALENs is complicated, the modification efficiency is high enough to achieve homoplasmy of multicopy mitochondrial genomes. Here I describe how to design mitoTALENs to select a target, construct a vector, and select the mitochondrial transformants.

Abstract 3099: The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal hematopoietic stem/progenitor cells to maintain stemness

Abstract Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a primary AML culture model arranged in a hierarchy with defined stem cells. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. In contrast, other metabolic enzymes including PFK1, FH, PKM2, GPI1, ENO1, CS, ACO2, and SDHA1, 2 were not detected in the nucleus of these cells. We also detected HK2 in the nucleus of AML cell lines as well as 7 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals in 8227 and NB4 leukemia cells. This increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Nuclear HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with 3'UTR shRNA. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation with ATRA, and decreased CD34+CD38- 8227 stem cells without changing basal proliferation. Nuclear HK2 was also higher in normal human hematopoietic stem cells and multipotent progenitor fractions and declined as the cells matured. Over-expression of nuclear HK2 in normal cord blood increased the primary and secondary engraftment into mice. Transgenic mice over-expressing nuclear HK2 driven by a Vav promoter had increased hematopoietic stem cells in the marrow and decreased monocytes and lymphocytes in the peripheral blood. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2 (D209A D657A) and observed increased clonogenic growth and inhibited differentiation on ATRA treatment, nuclear HK2 function is independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry identified proteins related to chromatin organization and regulation to interact with nuclear HK2. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Citation Format: Geethu Emily Thomas, Grace Egan, Laura Gracia Prat, Aaron Botham, Veronique Voisin, Elias Orouji, Jordan Chin, Boaz Nachmias, Kerstin B. Kaufmann, Neil Maclean, Rose Hurren, Marcela Gronda, Xiaoming Wang, Dilshad H. Khan, Rashim P. Singh, Andrea Arruda, Mark Minden, Gary D. Bader, John E. Dick, Aaron D. Schimmer. The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal hematopoietic stem/progenitor cells to maintain stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3099.

The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness

Abstract Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .

Bi-allelic HPDL Variants Cause a Neurodegenerative Disease Ranging from Neonatal Encephalopathy to Adolescent-Onset Spastic Paraplegia

We report bi-allelic pathogenic HPDL variants as a cause of a progressive, pediatric-onset spastic movement disorder with variable clinical presentation. The single-exon gene HPDL encodes a protein of unknown function with sequence similarity to 4-hydroxyphenylpyruvate dioxygenase. Exome sequencing studies in 13 families revealed bi-allelic HPDL variants in each of the 17 individuals affected with this clinically heterogeneous autosomal-recessive neurological disorder. HPDL levels were significantly reduced in fibroblast cell lines derived from more severely affected individuals, indicating the identified HPDL variants resulted in the loss of HPDL protein. Clinical presentation ranged from severe, neonatal-onset neurodevelopmental delay with neuroimaging findings resembling mitochondrial encephalopathy to milder manifestation of adolescent-onset, isolated hereditary spastic paraplegia. All affected individuals developed spasticity predominantly of the lower limbs over the course of the disease. We demonstrated through bioinformatic and cellular studies that HPDL has a mitochondrial localization signal and consequently localizes to mitochondria suggesting a putative role in mitochondrial metabolism. Taken together, these genetic, bioinformatic, and functional studies demonstrate HPDL is a mitochondrial protein, the loss of which causes a clinically variable form of pediatric-onset spastic movement disorder.

Impaired Female Reproductive Function in A Novel Estrogen Receptor Alpha Hypomorphic Mouse Model

IntroductionEstrogen signaling plays an important role in the regulation of both energy homeostasis and reproductive function. Human and animal studies indicate that the metabolic actions of estrogen signaling are primarily mediated by estrogen receptor alpha (ERα). Both human and mouse ERα genes show remarkable heterogeneity due to complicated alternative splicing, which results in a wide variety of variant transcripts with different protein products or a common protein. However, functional significance of this ERα genomic complexity has not been well documented. Here we generated a novel mouse model in which ERα expression was differentially affected in various tissues, and examined their body weight change and reproduction.MethodUsing CRISPR/Cas9 technology, we introduced a loxP‐flanked sequence containing En2 splice acceptor, a green fluorescent marker with mitochondrial localization signal and DNA translation stop signals before the first coding exon of mouse ERα gene for the potential restoration of the ERα expression.ResultsERα protein expressions were significantly reduced in pituitary (~37%) and ovary (~67%), but not in brain, of the homozygous mutant female compared to those of controls, indicating that unknown elements important for ERα tissue‐specific expression were affected. In contrast to body weight gain observed in previous studies of ERα knockout female mice, our hypomorphic mutant females did not show significant body weight gain. On the other hand, virginal smears from the mutant females rarely showed typical estrus stage. Uterine wet weight was significantly lower in the mutant female mice compared to the controls, indicating abnormal reproductive system. Continuous mating study further confirmed delayed pregnancy and inability to have alive newborns in the mutant female mice.ConclusionEstrogen receptor alpha protein expression varies among tissues in terms of the level and pattern of splicing variants. Splicing variant‐specific ERα functions are not fully elucidated. Our results suggest that a particular variant affected in our mouse model is less critical for proper functioning of systems involved in estrogen‐regulated energy metabolism, but indispensable for some aspects of female reproductive function.

The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness

Hematopoietic cells are arranged in a hierarchy where mature blood cells arise from stem and progenitor precursors. AML is also hierarchical with differentiated blasts arising from leukemic stem/progenitor cells. Recent studies show that metabolites can affect epigenetic marks; however, it is unknown whether metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we discovered that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. Metabolic enzymes that localize to nucleus of stem cells were identified by evaluating stem and bulk fractions of OCI-AML-8227 leukemia cells, which are arranged in a hierarchy with functionally defined stem cells. We separated OCI-AML-8227 cells into CD34+38- and CD34-38+ populations by FACS and prepared nuclear and cytoplasmic lysates. Immunoblotting of the lysates revealed that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Enolase1, Aconitase2, and Succinate Dehydrogenase A & B, were not detected in the nuclear lysates. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, initiating glycolysis. We confirmed nuclear HK2 in OCI-AML-8227 stem cells by confocal microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K563, and MV411) and in 7 of 9 primary AML samples. We FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (Monocytes, Granulocytes, B, T, and NK) cells. The localization of HK2 in these cells was analysed and quantified by immunofluorescence. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as cells matured. Next, we explored mechanisms that regulate nuclear localization of HK2. AKT-mediated phosphorylation of HK2 promoted localization to mitochondria while inhibition of phosphorylation increased its nuclear levels. Moreover, the nuclear import of HK2 was dependent on IPO5, a member of b-importin family that imports protein to the nucleus; CRM1 was responsible for HK2 nuclear export. We tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We overexpressed HK2 tagged with nuclear localizing signals (PKKKRKV or PAAKRVKLD) in 8227 and NB4 leukemia cells. Selective overexpression of HK2 in the nucleus did not alter the rate of proliferation of the cells, however there was enhanced clonogenic growth and inhibition of retinoic acid-mediated cell differentiation. Conversely, we selectively reduced nuclear HK2 by expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 in AML cells did not alter growth rate, but did reduce clonogenic growth and increased differentiation after treatment with retinoic acid. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2. A top hit in our screen was Exonuclease 3'-5' domain containing 2 (EXD2), involved in DNA repair. Of note, DNA damage induces differentiation of AML cells. In 8227 cells, nuclear EXD2 was higher in the stem cell fraction compared to the bulk fraction. Moreover, knockdown of EXD2 reduced AML growth, clonogenic growth and decreased nuclear HK2 levels. Finally, nuclear HK2 overexpression conferred resistance to the PARP inhibitor, olaparib. In summary, we discovered that unphosphorylated HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells. Through mechanisms independent of its kinase function, nuclear HK2 maintains AML cells in their stem/progenitor state potentially by regulating DNA damage and repair. Thus, we define a new role for a mitochondrial enzyme in the regulation of stemness and differentiation. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy.

High-level production of linalool by engineered Saccharomyces cerevisiae harboring dual mevalonate pathways in mitochondria and cytoplasm

Linalool, a valuable monoterpene alcohol, is widely used in cosmetics and flavoring ingredients. However, its scalable production by microbial fermentation is not yet achieved. In this work, considerable increase in linalool production was obtained in Saccharomyces cerevisiae by dual metabolic engineering of the mevalonic acid (MVA) pathway in both mitochondria and cytoplasm. A farnesyl pyrophosphate synthase mutant ERG20F96W/N127W and a linalool synthase from Cinnamomum osmophloeum (CoLIS) were introduced and meanwhile the endogenous ERG20 was down-regulated to prevent the competitive loss of precursor. In addition, overexpression of the proteins of CoLIS and ERG20F96W/N127W and another copy of the same enzymes CoLIS/ERG20F96W/N127W with mitochondrial localization signal (MLS) were carried out to further pull the flux to linalool. Finally, a maximum linalool titer of 23.45 mg/L was obtained in a batch fermentation with sucrose as carbon source. This combinatorial engineering strategy may provide hints for biosynthesis of other monoterpenes.

In-Depth Bioinformatic Study of the CLDN16 Gene and Protein: Prediction of Subcellular Localization to Mitochondria.

Background and Objectives: The defects in the CLDN16 gene are a cause of primary hypomagnesemia (FHHNC), which is characterized by massive renal magnesium wasting, resulting in nephrocalcinosis and renal failure. The mutations occur throughout the gene’s coding region and can impact on intracellular trafficking of the protein or its paracellular pore forming function. To gain more understanding about the mechanisms by which CLDN16 mutations can induce FHHNC, we performed an in-depth computational analysis of the CLDN16 gene and protein, focusing specifically on the prediction of the latter’s subcellular localization. Materials and Methods: The complete nucleotide or amino acid sequence of CLDN16 in FASTA format was entered and processed in 14 databases. Results: One CpG island was identified. Twenty five promoters/enhancers were predicted. The CLDN16 interactome was found to consist of 20 genes, mainly involved in kidney diseases. No signal peptide cleavage site was identified. A probability of export to mitochondria equal to 0.9740 and a cleavable mitochondrial localization signal in the N terminal of the CLDN16 protein were predicted. The secondary structure prediction was visualized. Νo phosphorylation sites were identified within the CLDN16 protein region by applying DISPHOS to the functional class of transport. The KnotProt database did not predict any knot or slipknot in the protein structure of CLDN16. Seven putative miRNA binding sites within the 3’-UTR region of CLDN16 were identified. Conclusions: This is the first study to identify mitochondria as a probable cytoplasmic compartment for CLDN16 localization, thus providing new insights into the protein’s intracellular transport. The results relative to the CLDN16 interactome underline its role in renal pathophysiology and highlight the functional dependence of CLDNs-10, 14, 16, 19. The predictions pertaining to the miRNAs, promoters/enhancers and CpG islands of the CLDN16 gene indicate a strict regulation of its expression both transcriptionally and post-transcriptionally.

Conserved motifs in nuclear genes encoding predicted mitochondrial proteins in Trypanosoma cruzi.

Trypanosoma cruzi, the protozoan parasite that causes Chagas’ disease, exhibits peculiar biological features. Among them, the presence of a unique mitochondrion is remarkable. Even though the mitochondrial DNA constitutes up to 25% of total cellular DNA, the structure and functionality of the mitochondrion are dependent on the expression of the nuclear genome. As in other eukaryotes, specific peptide signals have been proposed to drive the mitochondrial localization of a subset of trypanosomatid proteins. However, there are mitochondrial proteins encoded in the nuclear genome that lack of a peptide signal. In other eukaryotes, alternative protein targeting to subcellular organelles via mRNA localization has also been recognized and specific mRNA localization towards the mitochondria has been described. With the aim of seeking for mitochondrial localization signals in T. cruzi, we developed a strategy to build a comprehensive database of nuclear genes encoding predicted mitochondrial proteins (MiNT) in the TriTryps (T. cruzi, T. brucei and L. major). We found that approximately 15% of their nuclear genome encodes mitochondrial products. In T. cruzi the MiNT database reaches 1438 genes and a conserved peptide signal, M(L/F) R (R/S) SS, named TryM-TaPe is found in 60% of these genes, suggesting that the canonical mRNA guidance mechanism is present. In addition, the search for compositional signals in the transcripts of T. cruzi MiNT genes produce a list, being worth to note a conserved non-translated element represented by the consensus sequence DARRVSG. Taking into account its reported interaction with the T. brucei TRRM3 protein which is enriched in the mitochondrial membrane fraction, we here suggest a putative zip code role for this element. Globally, here we provide an inventory of the mitochondrial proteins in T. cruzi and give evidence for the existence of both peptide and mRNA signals specific to nuclear encoded mitochondrial proteins.