Mitochondrial Subcellular Localization Research Articles

HSP70-mediated mitochondrial dynamics and autophagy represent a novel vulnerability in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is one of the deadliest forms of cancer with limited therapy options. Overexpression of the heat shock protein 70 (HSP70) is a hallmark of cancer that is strongly associated with aggressive disease and worse clinical outcomes. However, the underlying mechanisms by which HSP70 allows tumor cells to thrive under conditions of continuous stress have not been fully described. Here, we report that PDAC has the highest expression of HSP70 relative to normal tissue across all cancers analyzed. Furthermore, HSP70 expression is associated with tumor grade and is further enhanced in metastatic PDAC. We show that genetic or therapeutic ablation of HSP70 alters mitochondrial subcellular localization, impairs mitochondrial dynamics, and promotes mitochondrial swelling to induce apoptosis. Mechanistically, we find that targeting HSP70 suppresses the PTEN-induced kinase 1 (PINK1) mediated phosphorylation of dynamin-related protein 1 (DRP1). Treatment with the HSP70 inhibitor AP-4-139B was efficacious as a single agent in primary and metastatic mouse models of PDAC. In addition, we demonstrate that HSP70 inhibition promotes the AMP-activated protein kinase (AMPK) mediated phosphorylation of Beclin-1, a key regulator of autophagic flux. Accordingly, we find that the autophagy inhibitor hydroxychloroquine (HCQ) enhances the ability of AP-4-139B to mediate anti-tumor activity in vivo. Collectively, our results suggest that HSP70 is a multi-functional driver of tumorigenesis that orchestrates mitochondrial dynamics and autophagy. Moreover, these findings support the rationale for concurrent inhibition of HSP70 and autophagy as a novel therapeutic approach for HSP70-driven PDAC.

1593-P: Insulin-Degrading Enzyme (IDE) Regulates Glucagon Signalling and Mitochondrial Respiration in Hepatocytes

IDE is a ubiquitous metalloprotease with cytosolic and mitochondrial subcellular localization that degrades insulin and glucagon. People with T2D and diet-induced obese mice show lower hepatic IDE levels. We revealed a key role of IDE on the insulin-mediated repression of hepatic gluconeogenesis, but its function on glucagon-dependent activation of gluconeogenesis and mitochondrial respiration in hepatocytes remains completely unknown. Here, we aim to elucidate the role of IDE on glucagon signalling and its impact on gluconeogenesis and energy metabolism in hepatocytes. Liver homogenized and primary hepatocytes obtained from L-IDE-KO mice (deletion of IDE in liver) showed decreased expression of glucagon receptor (~60%), CREB protein (~40%), and lower phosphorylation of CREB (~50%) upon glucagon stimulation compared to controls. Similar results were found in AML12-shRNA-Ide cells, in which IDE protein levels were reduced by ~50%. Additionally, glucagon stimulation resulted in lower (~30%) cAMP levels and diminished phosphorylation of PKA substrates in AML12-shRNA-Ide. Surprisingly, these alterations in glucagon signalling paralleled with ~20-fold increases in the expression of the gluconeogenic genes G6p6 and Pck1. Of note, basal and uncoupler-stimulated respiration increased ~4-fold in AML12-shRNA-Ide in parallel with a ~2-fold increment of mitochondrial and glycolytic ATP production. Finally, similar mitochondrial phenotype was found in human hepatocytes lacking IDE (HepG2-IDE-KO cells), which exhibited higher FoxO1 levels and fragmented mitochondria. The effects on mitochondrial respiration were independent of IDE's proteolytic activity. In summary, reduced IDE expression in hepatocytes has a deleterious effect on glucagon signalling leading to up-regulated gluconeogenesis and mitochondrial respiration. We conclude that IDE is a mechanistic link to couple hepatic gluconeogenesis with mitochondrial energy production. Disclosure C.M. González-Casimiro: None. P. Cámara-Torres: None. B. Merino: None. J. Santo-Domingo: None. M.A. de la Fuente: None. A. Alonso: None. I. Cozar-Castellano: None. G. Perdomo: None. Funding Ministerio de Ciencia e Innovación-Spain (PID2019-110496RB-C22)

SUMOylation of SYNJ2BP-COX16 promotes breast cancer progression through DRP1-mediated mitochondrial fission

Treatments targeting oncogenic fusion proteins are notable examples of successful drug development. Abnormal splicing of genes resulting in fusion proteins is a critical driver of various tumors, but the underlying mechanism remains poorly understood. Here, we show that SUMOylation of the fusion protein Synaptojanin 2 binding protein-Cytochrome-c oxidase 16 (SYNJ2BP-COX16) at K107 induces mitochondrial fission in breast cancer and that the K107 site regulates SYNJ2BP-COX16 mitochondrial subcellular localization. Compared with a non-SUMOylated K107R mutant, wild-type SYNJ2BP-COX16 contributed to breast cancer cell proliferation and metastasis in vivo and in vitro by increasing adenosine triphosphate (ATP) production and cytochrome-c oxidase (COX) activity. SUMOylated SYNJ2BP-COX16 recruits dynamin-related protein 1 (DRP1) to the mitochondria to promote ubiquitin-conjugating enzyme 9 (UBC9) binding to DRP1, enhance SUMOylation of DRP1 and phosphorylation of DRP1 at S616, and then induce mitochondrial fission. Moreover, Mdivi-1, an inhibitor of DRP1 phosphorylation, decreased the localization of DRP1 in mitochondria, and prevents SYNJ2BP-COX16 induced mitochondrial fission, cell proliferation and metastasis. Based on these data, SYNJ2BP-COX16 promotes breast cancer progression through the phosphorylation of DRP1 and subsequent induction of mitochondrial fission, indicating that SUMOylation at the K107 residue of SYNJ2BP-COX16 is a novel potential treatment target for breast cancer.

Inflammation-induced PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons

BackgroundDiseases and disorders with a chronic neuroinflammatory component are often linked with changes in brain metabolism. Among neurodegenerative disorders, people living with human immunodeficiency virus (HIV) and Alzheimer’s disease (AD) are particularly vulnerable to metabolic disturbances, but the mechanistic connections of inflammation, neurodegeneration and bioenergetic deficits in the central nervous system (CNS) are poorly defined. The particularly interesting new cysteine histidine-rich-protein (PINCH) is nearly undetectable in healthy mature neurons, but is robustly expressed in tauopathy-associated neurodegenerative diseases including HIV infection and AD. Although robust PINCH expression has been reported in neurons in the brains of patients with HIV and AD, the molecular mechanisms and cellular consequences of increased PINCH expression in CNS disease remain largely unknown.MethodsWe investigated the regulatory mechanisms responsible for PINCH protein-mediated changes in bioenergetics, mitochondrial subcellular localization and bioenergetic deficits in neurons exposed to physiological levels of TNFα or the HIV protein Tat. Changes in the PINCH-ILK-Parvin (PIP) complex association with cofilin and TESK1 were assessed to identify factors responsible for actin depolymerization and mitochondrial mislocalization. Lentiviral and pharmacological inhibition experiments were conducted to confirm PINCH specificity and to reinstate proper protein-protein complex communication.ResultsWe identified MEF2A as the PINCH transcription factor in neuroinflammation and determined the biological consequences of increased PINCH in neurons. TNFα-mediated activation of MEF2A via increased cellular calcium induced PINCH, leading to disruption of the PIP ternary complex, cofilin activation by TESK1 inactivation, and actin depolymerization. The disruption of actin led to perinuclear mislocalization of mitochondria by destabilizing the kinesin-dependent mitochondrial transport machinery, resulting in impaired neuronal metabolism. Blocking TNFα-induced PINCH expression preserved mitochondrial localization and maintained metabolic functioning.ConclusionsThis study reported for the first time the mechanistic and biological consequences of PINCH expression in CNS neurons in diseases with a chronic neuroinflammation component. Our findings point to the maintenance of PINCH at normal physiological levels as a potential new therapeutic target for neurodegenerative diseases with impaired metabolisms.

Mitochondrial sub-cellular localization of cAMP-specific phosphodiesterase 8A in ovarian follicular cells

Cyclic adenosine monophosphate (cAMP) is a ubiquitous secondary messenger that plays a central role in endocrine tissue function, particularly in the synthesis of steroid hormones. The intracellular concentration of cAMP is regulated through its synthesis by cyclases and its degradation by cyclic nucleotide phosphodiesterases (PDEs). Although the expression and activity of PDEs impact the specificity and the amplitude of the cAMP response, it is becoming increasingly clear that the sub-cellular localization of PDE emphasizes the spatial regulation of the cell signalling processes that are essential for normal cellular function. We first examined the expression of PDE8A in porcine ovarian cells. PDE8A is expressed in granulosa cells, cumulus cells and oocytes. Second, we assessed the mitochondrial sub-cellular localization of PDE8A. Using western blotting with isolated mitochondrial fractions from granulosa cells and cumulus-oocyte complexes revealed immuno-reactive bands. PDE assay of isolated mitochondrial fractions from granulosa cells measured specific PDE8 cAMP-PDE activity as PF-04957325-sensitive. The immune-reactive PDE8A signal and MitoTracker labelling co-localized supporting mitochondrial sub-cellular localization of PDE8A, which was confirmed using immuno-electron microscopy. Finally, the effect of PDE8 on progesterone production was assessed during the in-vitro maturation of cumulus-oocyte complexes. Using PF-04957325, we observed a significant increase (P < 0.05) in progesterone secretion with follicle-stimulating hormone (FSH). Active mitochondria stained with MitoTracker orange CMTMRos were also increased by the specific PDE8 inhibitor supporting its functional regulation. In conclusion, we propose the occurrence of mitochondrial sub-cellular localization of PDE8A in porcine granulosa cells and cumulus cells. This suggests that there is potential for new strategies for ovarian stimulation and artificial reproductive technologies, as well as the possibility for using new media to improve the quality of oocytes.

Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson's Disease.

Parkinson’s disease (PD) is the second most frequent neurodegenerative disease worldwide and the availability of early biomarkers and novel biotargets represents an urgent medical need. The main pathogenetic hallmark of PD is the specific loss of nigral dopaminergic neurons, in which mitochondrial dysfunction plays a crucial role. Mitochondrial proteases are central to the maintenance of healthy mitochondria and they have recently emerged as drug targets. However, an exhaustive characterization of these enzymes and their targets is still lacking, due to difficulties in analyzing proteolytic fragments by bottom-up proteomics approaches. Here, we propose the “mitochondrial dimethylation-TAILS” strategy, which combines the isolation of mitochondria with the enrichment of N-terminal peptides to analyze the mitochondrial N-terminome. We applied this method in a cellular model of altered dopamine homeostasis in neuroblastoma SH-SY5Y cells, which recapitulates early steps of PD pathogenesis. The main aim was to identify candidate mitochondrial proteases aberrantly activated by dopamine dysregulation and their cleaved targets. The proposed degradomics workflow was able to improve the identification of mitochondrial proteins if compared to classical shotgun analysis. In detail, 40% coverage of the mitochondrial proteome was obtained, the sequences of the transit peptides of two mitochondrial proteins were unveiled, and a consensus cleavage sequence for proteases involved in the processing of mitochondrial proteins was depicted. Mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD013900. Moreover, sixty-one N-terminal peptides whose levels were affected by dopamine treatment were identified. By an in-depth analysis of the proteolytic peptides included in this list, eleven mitochondrial proteins showed altered proteolytic processing. One of these proteins (i.e., the 39S ribosomal protein L49 – MRPL49) was cleaved by the neprilysin protease, already exploited in clinics as a biotarget. We eventually demonstrated a mitochondrial subcellular localization of neprilysin in human cells for the first time. Collectively, these results shed new light on mitochondrial dysfunction linked to dopamine imbalance in PD and opened up the possibility to explore the mitochondrial targets of neprilysin as candidate biomarkers.

PO-243 Uncoupling FOXO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy

IntroductionWhile aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrial functions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is a key determinant of cancer cell homeostasis, playing a dual role in survival/death response. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells upon nutrient shortage.Material and methodsAfter extensive characterisation of mitochondrial FoxO3A function in vitro in several cell lines and tumours, we generated FoxO3A-knockout cancer cells with the CRISPR/Cas9 system and reconstituted FoxO3A expression with wild-type or mutant vectors.Results and discussionsHere we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression of the mitochondrial genome to support mitochondrial metabolism and cell survival. Using FoxO3A-/- cancer cells generated with the CRISPR/Cas9 genome editing system and reconstituted with FoxO3A mutants being impaired in their nuclear or mitochondrial subcellular localization, we show that mitochondrial FoxO3A promotes survival in response to metabolic stress.In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into the mitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required for apoptosis induction by metformin.ConclusionElucidation of FoxO3A mitochondrial vs. nuclear functions in cancer cell homeostasis might help devise novel personalised therapeutic strategies to selectively disable FoxO3A pro-survival activity and manipulate cellular metabolism to counteract cancer initiation and progression.

Uncoupling FoxO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy

While aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrial functions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is a key determinant of cancer cell homeostasis, playing a dual role in survival/death response to metabolic stress and cancer therapeutics. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells upon nutrient shortage. Here, we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression of the mitochondrial genome to support mitochondrial metabolism. Using FoxO3A−/− cancer cells generated with the CRISPR/Cas9 genome editing system and reconstituted with FoxO3A mutants being impaired in their nuclear or mitochondrial subcellular localization, we show that mitochondrial FoxO3A promotes survival in response to metabolic stress. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into the mitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required for apoptosis induction by metformin. Elucidation of FoxO3A mitochondrial vs. nuclear functions in cancer cell homeostasis might help devise novel therapeutic strategies to selectively disable FoxO3A prosurvival activity.

Proteomic profiling of neuronal mitochondria reveals modulators of synaptic architecture

BackgroundNeurons are highly polarized cells consisting of three distinct functional domains: the cell body (and associated dendrites), the axon and the synapse. Previously, it was believed that the clinical phenotypes of neurodegenerative diseases were caused by the loss of entire neurons, however it has recently become apparent that these neuronal sub-compartments can degenerate independently, with synapses being particularly vulnerable to a broad range of stimuli. Whilst the properties governing the differential degenerative mechanisms remain unknown, mitochondria consistently appear in the literature, suggesting these somewhat promiscuous organelles may play a role in affecting synaptic stability. Synaptic and non-synaptic mitochondrial subpools are known to have different enzymatic properties (first demonstrated by Lai et al., 1977). However, the molecular basis underpinning these alterations, and their effects on morphology, has not been well documented.MethodsThe current study has employed electron microscopy, label-free proteomics and in silico analyses to characterize the morphological and biochemical properties of discrete sub-populations of mitochondria. The physiological relevance of these findings was confirmed in-vivo using a molecular genetic approach at the Drosophila neuromuscular junction.ResultsHere, we demonstrate that mitochondria at the synaptic terminal are indeed morphologically different to non-synaptic mitochondria, in both rodents and human patients. Furthermore, generation of proteomic profiles reveals distinct molecular fingerprints – highlighting that the properties of complex I may represent an important specialisation of synaptic mitochondria. Evidence also suggests that at least 30% of the mitochondrial enzymatic activity differences previously reported can be accounted for by protein abundance. Finally, we demonstrate that the molecular differences between discrete mitochondrial sub-populations are capable of selectively influencing synaptic morphology in-vivo. We offer several novel mitochondrial candidates that have the propensity to significantly alter the synaptic architecture in-vivo.ConclusionsOur study demonstrates discrete proteomic profiles exist dependent upon mitochondrial subcellular localization and selective alteration of intrinsic mitochondrial proteins alters synaptic morphology in-vivo.

Antitumor effect of sinoporphyrin sodium-mediated photodynamic therapy on human esophageal cancer Eca-109 cells.

The aim of this study was to evaluate the photodynamic effect of Sinoporphyrin sodium (DVDMS). In this study, Eca-109 cells were treated with DVDMS (5 μg mL(-1)) and subjected to photodynamic therapy (PDT). The uptake and subcellular localization of DVDMS were monitored by flow cytometry and confocal microscopy. The phototoxicity of DVDMS was studied by MTT assay. The morphological changes were observed by scanning electron microscopy (SEM). DNA damage, reactive oxygen species (ROS) generation and mitochondria membrane potential (MMP) changes were analyzed by flow cytometry. Studies demonstrated maximal uptake of DVDMS occurred within 3 h, with a mitochondrial subcellular localization. MTT assays displayed that DVDMS could be effectively activated by light and the phototoxicity was much higher than photofrin under the same conditions. In addition, SEM observation indicated that cells were seriously damaged after PDT treatment. Furthermore, activation of DVDMS resulted in significant increases in ROS production. The generated ROS played an important role in the phototoxicity of DVDMS. DVDMS-mediated PDT (DVDMS-PDT) also induced DNA damage and MMP loss. It is demonstrated that DVDMS-mediated PDT is an effective approach on cell proliferation inhibition of Eca-109 cells.

Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alpha-synuclein.

The specific and effective α-synuclein RNA interference (RNAi) plasmids, and the α-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 (HEK293) cells using the lipofectamine method. Using an inverted fluorescence microscope, α-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type α-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies (Lewy body-like inclusions) in the cytoplasm of some cells transfected with α-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type α-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type α-synuclein in mitochondria.

A TSPO-related protein localizes to the early secretory pathway in Arabidopsis, but is targeted to mitochondria when expressed in yeast

AtTSPO is a TspO/MBR domain-protein potentially involved in multiple stress regulation in Arabidopsis. As in most angiosperms, AtTSPO is encoded by a single, intronless gene. Expression of AtTSPO is tightly regulated both at the transcriptional and post-translational levels. It has been shown previously that overexpression of AtTSPO in plant cell can be detrimental, and the protein was detected in the endoplasmic reticulum (ER) and Golgi stacks, contrasting with previous findings and suggesting a mitochondrial subcellular localization for this protein. To ascertain these findings, immunocytochemistry and ABA induction were used to demonstrate that, in plant cells, physiological levels of AtTSPO colocalized with AtArf1, a mainly Golgi-localized protein in plant cells. In addition, fluorescent protein-tagged AtTSPO was targeted to the secretory pathway and did not colocalize with MitoTracker-labelled mitochondria. These results suggest that the polytopic membrane protein AtTSPO is cotranslationally targeted to the ER in plant cells and accumulates in the Trans-Golgi Network. Heterologous expression of AtTSPO in Saccharomyces cerevisiae, yeast devoid of TSPO-related protein, resulted in growth defects. However, subcellular fractionation and immunoprecipitation experiments showed that AtTSPO was targeted to mitochondria where it colocalized and interacted with the outer mitochondrial membrane porin VDAC1p, reminiscent of the subcellular localization and activity of mammalian translocator protein 18 kDa TSPO. The evolutionarily divergent AtTSPO appears therefore to be switching its sorting mode in a species-dependent manner, an uncommon peculiarity for a polytopic membrane protein in eukaryotic cells. These results are discussed in relation to the recognition and organelle targeting mechanisms of polytopic membrane proteins in eukaryotic cells.

Sonodynamic and photodynamic mechanisms of action of the novel hypocrellin sonosensitizer, SL017: mitochondrial cell death is attenuated by 11, 12-epoxyeicosatrienoic acid

Development of sonosensitizers for sonodynamic therapy (SDT) which selectively target abnormal cells can limit undesired side effects in chemotherapeutic applications. Hypocrellin-B (HB) derivatives are low molecular weight compounds which belong to the perylenequinone family of photosensitizing and sonosensitizing compounds. In this study, we investigate the cytotoxic mechanisms of a novel HB-derived photo- and sonosensitizer, SL017. Human fibroblast WI-38 cells were treated with SL017 (0μM, 0.1μM or 10μM) and subjected to photodynamic therapy (PDT) or SDT. Studies demonstrate that maximal uptake of SL017 occurs within 30min, with a mitochondrial subcellular localization. Activation of SL017 by either visible light or ultrasound resulted in significant increases in reactive oxygen species (ROS) production as measured by CM-H2-DCFDA (5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester). Co-administration of the antioxidant, ascorbic acid, attenuated ROS production. Low concentrations of SL017 (100nM) induced a rapid (<90s) loss of mitochondrial membrane potential (ΔΨm). Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid (AA) involved in maintaining homeostasis and protection against cell injury, were able to attenuate loss of ΔΨm, however ascorbic acid was not. SL017 treatment resulted in increased mitochondrial fragmentation which followed loss of ΔΨm. Our studies demonstrate that SL017 targets mitochondria, triggering collapse of mitochondrial membrane potential, generates ROS and subsequently results in mitochondrial fragmentation.

Clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin.

Parkinson's disease has been linked to altered mitochondrial function. Mutations in parkin (park), the Drosophila ortholog of a human gene that is responsible for many familial cases of Parkinson's disease, shorten life span, abolish fertility and disrupt mitochondrial structure. However, the role played by Park in mitochondrial function remains unclear. Here, we describe a novel Drosophila gene, clueless (clu), which encodes a highly conserved tetratricopeptide repeat protein that is related closely to the CluA protein of Dictyostelium, Clu1 of Saccharomyces cerevisiae and to similar proteins in diverse metazoan eukaryotes from Arabidopsis to humans. Like its orthologs, loss of Drosophila clu causes mitochondria to cluster within cells. We find that strong clu mutations resemble park mutations in their effects on mitochondrial function and that the two genes interact genetically. Conversely, mitochondria in park homozygotes become highly clustered. We propose that Clu functions in a novel pathway that positions mitochondria within the cell based on their physiological state. Disruption of the Clu pathway may enhance oxidative damage, alter gene expression, cause mitochondria to cluster at microtubule plus ends, and lead eventually to mitochondrial failure.

The carnitine acetyltransferase gene (CRAT): A characterization of porcine transcripts with insights into the 5’-end variants of mammalian transcripts and their possible sub-cellular localization

Carnitine acetyltransferase (CRAT) is an important enzyme for energy homeostasis and fat metabolism. We characterized the predicted full length cDNA sequence of the porcine CRAT gene. Its structure is very similar to that in humans with respect to the size and organization of the 14 exons. We demonstrated the existence of a porcine alternative transcript resulting from a partial intron-retention at the 5’ end of exon 2. To perform a comparison of the 5’ end variants of the mammalian CRAT gene, we analyzed the Genbank data, and here we propose a new 5’ variant for dog, rat and mouse. In contrast to other mammals where this variant encodes a shorter protein (−21 aa in human, mouse and rat, and −14 aa in dog), the pig variant encodes for a longer protein (+18 aa). In all mammalian species, variant 1 has a high probability of a preferential mitochondrial sub-cellular localization. Nevertheless, it is not evident, in particular in porcine and dog species, that the second variant is associated with a different sub-cellular specificity.

Expression of the scaffolding PDZ protein glutaminase‐interacting protein in mammalian brain

A human brain cDNA clone coding for a novel PDZ-domain protein of 124 amino acids was previously isolated in our laboratory. The protein was termed glutaminase-interacting protein (GIP), because it interacts with the C-terminal region of the human L-type glutaminase (LGA). The pattern of expression and functions of GIP in brain are completely unknown, so its significance remains undefined. Here we describe the expression of GIP mRNA and protein in mammalian brain. Northern blot analysis revealed that GIP mRNA was ubiquitous in most regions of human brain but was particularly abundant in spinal cord. The presence of the protein in rat and monkey brain was studied at the regional, cellular, and subcellular level by immunocytochemistry. The protein was found to be present in both neurons and astrocytes, with a cytosolic and mitochondrial subcellular localization. Double immunofluorescence labeling with anti-GIP and anti-LGA antibodies using confocal microscopy revealed colocalization of both proteins in astrocyte cell processes and their perivascular end feet. Electron microscopy of rat brain neurons revealed GIP immunoreactivity concentrated also in the nuclear envelope and the plasma membrane. The multiple locations for GIP in mammalian brain are in agreement with known protein interaction partners reported for this PDZ protein. The findings presented here support a role of GIP as an important scaffold in both astrocytes and neurons and point toward astrocytic processes and perivascular end feet as plausible anatomical substrates for interaction with glutaminase.

In vitro phototoxicity of glycoconjugated porphyrins and chlorins in colorectal adenocarcinoma (HT29) and retinoblastoma (Y79) cell lines

Retinoblastoma is the most common malignant intraocular tumor in children. The current treatment gives a good vital prognostic but there are several drawbacks to the arsenal of "classical antitumoral" therapies. Photodynamic therapy (PDT) could be an exciting non-toxic and non-mutagenic alternative protocol. In this paper, we report about the screening of the in vitro photocytotoxicity of hydrophenylporphyrins and chlorins and their glycoconjugated derivatives in a human retinoblastoma cell line (Y79) and for comparison in a colorectal adenocarcinoma cell line (HT29). Despite lower photodynamic activity than that observed for hydroxylated photosensitizers, in particular Foscan(®) glycoconjugated derivatives display phototoxicity (IC50 2.4-0.05μM ±10%) against Y79 cells with examples of significant intrinsic cytotoxicity. Amongst them the triglucosyl porphyrin 10 is highly photocytotoxic (IC50 0.9μM ±10%) but is fully devoid of cytotoxicity (IC50>15μM). The photoactivity is highly modulated by the presence of a diethyleneglycol spacer between the chromophore and the glycoside (compounds 14-17, IC50 0.5, 0.6, 0.05 and 0.35μM ±10%) and by the anomeric configuration of the sugar (compound 15 and 17, IC50 0.6 and 0.05μM ±10% respectively). One of the main problems for the use of Foscan(®) is its poor solubility which might be improved by glycoconjugation. Moreover Foscan has been shown to induce necrosis after PDT leading to a possible ulceration of surrounding tissues unsuitable for a conservative treatment. A preferential mitochondrial subcellular localization which has been previously reported for some glycoconjugated photosensitizers could enhance the contribution of apoptosis process. Tri-α-O-galactosyl porphyrin 16 is a better candidate than Foscan(®) for a clinical application of PDT for a conservative therapy of retinoblastoma.

Manganese superoxide dismutase based phylogeny of pathogenic fungi

Superoxide dismutases (SODs), which provide protection against oxidative stress, exhibit an essential role for fungal cell survival, especially during host invasion. Here, 20 partial SOD sequences from 19 pathogenic fungi were determined and aligned with 43 homologous fungal sequences from databases. All sequences encoded tetrameric manganese (Mn)-containing SODs showing predicted cytosolic or mitochondrial subcellular localization. Numerous fungi possessed both cytosolic and mitochondrial MnSODs in addition to the mainly cytosolic copper/zinc isozyme. Moreover, MnSOD sequence variability was higher than SSU rRNA and similar to ITS rRNA, suggesting MnSOD could be used to identify closely related fungal species. MnSOD- and SSU rRNA-based phylogenetic trees were constructed and compared. Despite a more complex topology of the MnSOD tree, due to several gene duplication events, all the classic fungal classes and orders were recovered. A salient point was the existence of two paralogous cytosolic and mitochondrial MnSODs in some Ascomycota. A hypothetical evolutionary scenario with an early gene duplication of the “mitochondrial” gene is proposed.

Cellular expression and alternative splicing of SLC25A23, a member of the mitochondrial Ca 2+-dependent solute carrier gene family

The transport of metabolites across the inner mitochondrial membrane is mediated by a large superfamily of mitochondrial solute carrier (MSC) proteins. A novel human member of the MSC gene family named SLC25A23, with homologs in mammalian and non-mammalian species has been recently identified together with two close paralogs, SLC25A24 and SLC25A25. These genes encode the human isoforms of the ATP-Mg/Pi carrier described in whole mitochondria. We report here the cellular expression and alternative splicing of SLC25A23. The gene encodes a 468 amino acids polypeptide, named SCaMC-3, with a bipartite structure typical of calcium-binding mitochondrial solute carrier (CaMSC) proteins. The amino-terminal portion harbors three canonical EF-hand calcium-binding domains while the carboxyl-terminal portion of SCaMC-3 has the characteristic features of the MSC superfamily. Northern blot analysis reveals the presence of the transcript in brain, heart, skeletal muscle, liver and small intestine. The SLC25A23 gene undergoes alternative splicing suggesting a modular nature of the encoded product. Three out of four putative protein isoforms lack a significant portion of the third mitochondrial carrier signature. The most common SCaMC-3 isoform shows a mitochondrial subcellular localization when transfected in HeLa cells and is able to bind calcium by Ca 2+-dependent mobility shift assays. We believe that our study will contribute to a better knowledge of this family of mitochondrial carriers.

The Nucleoside Diphosphate Kinase Activity of DRnm23 Is Not Required for Inhibition of Differentiation and Induction of Apoptosis in 32Dcl3 Myeloid Precursor Cells

DRnm23 belongs to a multigene family which includes nm23-H1, the first bona fide metastasis suppressor gene, nm23-H2, nm23-H4, and nm23-H5. Like nm23-H1, nm23-H2, and nm23-H4, DRnm23 possesses nucleoside diphosphate kinase (NDPK) activity. Upon overexpression in myeloid precursor 32Dcl3 cells, DRnm23 inhibits granulocytic differentiation and promotes apoptosis. Two specific mutants of DRnm23 (H134Q and S136P), at residues required for the NDPK activity, inhibit differentiation and promote apoptosis of 32Dcl3 cells. By contrast, substitution of serine 61 with proline (S61P) or deletion of the RGD domain (ΔRGD) abrogates the effects of wild-type DRnm23. Like wild-type DRnm23, all four mutants show a predominantly mitochondrial subcellular localization. These studies indicate that the enzymatic activity of DRnm23 is not required for the effects observed in 32Dcl3 cells. Moreover, the inability of the S61P and ΔRGD DRnm23 mutants to inhibit differentiation and promote apoptosis may be due to defective protein–protein interactions at the mitochondria, the predominant site of DRnm23 subcellular localization.