Mitochondrial Membrane Receptor Research Articles

Cadmium exposure activates mitophagy through downregulating thyroid hormone receptor/PGC1α signal in preeclampsia

Gestational cadmium exposure increases the risk of preeclampsia. Placenta mitophagy was activated in preeclampsia. The aim of present study was to explore the mechanism of cadmium-induced mitophagy activation and its association with preeclampsia. Mitophagy markers expression levels were detected by quantitative real-time PCR, Western blot, immunofluorescence and immunochemistry in preeclampsia placenta. JEG3 cells were treated with CdCl2, iopanoic acid (IOP), 3-methyladenine and PGC1α SiRNA to verify mechanism of cadmium-induced mitophagy. Mitophagy marker LC3BII/I and P62 expression were increased and mitochondrial membrane receptor protein TOM20 and FUNDC1 expression were decreased in preeclampsia placenta as compared with that in normotension control. Mitophagy marker LC3BII/I and P62 expression were increased and TOM20 and FUNDC1 expression was decreased in CdCl2-treated JEG3 cells. Meanwhile, mitochondrial biogenesis regulator, PGC1α expression was decreased in preeclampsia and CdCl2-treated JEG3 cells. The expressions of LC3B and P62 were increased and the expressions of TOM20, FUNDC1 and PGC1α were decreased in IOP-treated cell. PGC1α SiRNA transfection led to increased expression of LC3BII/I and P62 and decreased expression of TOM20 and FUNDC1. The expression of sFlt1 was increased in preeclampsia placenta, CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. 3-methyladenine treatment protected the increased expression of sFlt1 in CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. Meanwhile, co-treatment of cadmium and IOP or PGC1αSiRNA led to a reduce expressions of OPA1, MFN1, MFN2 and FUNDC1 as compared to cadmium-treated, IOP-treated and PGC1α SiRNA-treated cells. These results elucidated that maternal cadmium exposure activated placenta mitophagy through downregulation of thyroid hormone receptor signal mediated decreased expression of PGC1α and was associated with the occurrence of preeclampsia.

Mitophagy in neurodegenerative disease pathogenesis.

Mitochondria are critical cellular energy resources and are central to the life of the neuron. Mitophagy selectively clears damaged or dysfunctional mitochondria through autophagic machinery to maintain mitochondrial quality control and homeostasis. Mature neurons are postmitotic and consume substantial energy, thus require highly efficient mitophagy pathways to turn over damaged or dysfunctional mitochondria. Recent evidence indicates that mitophagy is pivotal to the pathogenesis of neurological diseases. However, more work is needed to study mitophagy pathway components as potential therapeutic targets. In this review, we briefly discuss the characteristics of nonselective autophagy and selective autophagy, including ERphagy, aggrephagy, and mitophagy. We then introduce the mechanisms of Parkin-dependent and Parkin-independent mitophagy pathways under physiological conditions. Next, we summarize the diverse repertoire of mitochondrial membrane receptors and phospholipids that mediate mitophagy. Importantly, we review the critical role of mitophagy in the pathogenesis of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Last, we discuss recent studies considering mitophagy as a potential therapeutic target for treating neurodegenerative diseases. Together, our review may provide novel views to better understand the roles of mitophagy in neurodegenerative disease pathogenesis.

UBQLN2 and HSP70 participate in Parkin-mediated mitophagy by facilitating outer mitochondrial membrane rupture.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two aging-related neurodegenerative diseases that share common key features, including aggregation of pathogenic proteins, dysfunction of mitochondria, and impairment of autophagy. Mutations in ubiquilin 2 (UBQLN2), a shuttle protein in the ubiquitin-proteasome system (UPS), can cause ALS/FTD, but the mechanism underlying UBQLN2-mediated pathogenesis is still uncertain. Recent studies indicate that mitophagy, a selective form of autophagy which is crucial for mitochondrial quality control, is tightly associated with neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and ALS. In this study, we show that after Parkin-dependent ubiquitination of damaged mitochondria, UBQLN2 is recruited to poly-ubiquitinated mitochondria through the UBA domain. UBQLN2 cooperates with the chaperone HSP70 to promote UPS-driven degradation of outer mitochondrial membrane (OMM) proteins. The resulting rupture of the OMM triggers the autophagosomal recognition of the inner mitochondrial membrane receptor PHB2. UBQLN2 is required for Parkin-mediated mitophagy and neuronal survival upon mitochondrial damage, and the ALS/FTD pathogenic mutations in UBQLN2 impair mitophagy in primary cultured neurons. Taken together, our findings link dysfunctional mitophagy to UBQLN2-mediated neurodegeneration.

Computational pharmacology and computational chemistry of 4-hydroxyisoleucine: Physicochemical, pharmacokinetic, and DFT-based approaches.

Computational pharmacology and chemistry of drug-like properties along with pharmacokinetic studies have made it more amenable to decide or predict a potential drug candidate. 4-Hydroxyisoleucine is a pharmacologically active natural product with prominent antidiabetic properties. In this study, ADMETLab 2.0 was used to determine its important drug-related properties. 4-Hydroxyisoleucine is compliant with important drug-like physicochemical properties and pharma giants' drug-ability rules like Lipinski's, Pfizer, and GlaxoSmithKline (GSK) rules. Pharmacokinetically, it has been predicted to have satisfactory cell permeability. Blood-brain barrier permeation may add central nervous system (CNS) effects, while a very slight probability of being CYP2C9 substrate exists. None of the well-known toxicities were predicted in silico, being congruent with wet lab results, except for a "very slight risk" for respiratory toxicity predicted. The molecule is non ecotoxic as analyzed with common indicators such as bioconcentration and LC50 for fathead minnow and daphnia magna. The toxicity parameters identified 4-hydroxyisoleucine as non-toxic to androgen receptors, PPAR-γ, mitochondrial membrane receptor, heat shock element, and p53. However, out of seven parameters, not even a single toxicophore was found. The density functional theory (DFT) study provided support to the findings obtained from drug-like property predictions. Hence, it is a very logical approach to proceed further with a detailed pharmacokinetics and drug development process for 4-hydroxyisoleucine.

Functional role and molecular mechanisms underlying prohibitin2 in platelet mitophagy and activation.

Platelet mitophagy is a major pathway involved in the clearance of injured mitochondria during hemostasis and thrombosis. Prohibitin 2 (PHB2) has recently emerged as an inner mitochondrial membrane receptor involved in mitophagy. However, the mechanisms underlying PHB2-mediated platelet mitophagy and activation are not completely understood. PHB2 is a highly conserved inner mitochondrial membrane protein that regulates mitochondrial assembly and function due to its unique localization on the mitochondrial membrane. The present study aimed to investigate the role and mechanism underlying PHB2 in platelet mitophagy and activation. Phorbol-12-myristate-13-acetate (PMA) was used to induce MEG-01 cells maturation and differentiate into platelets following PHB2 knockdown. Cell Counting Kit-8 assays were performed to examine platelet viability. Flow cytometry was performed to assess platelet mitochondrial membrane potential. RT-qPCR and western blotting were conducted to measure mRNA and protein expression levels, respectively. Subsequently, platelets were exposed to CCCP and the role of PHB2 was assessed. The results of the present study identified a crucial role for PHB2 in platelet mitophagy and activation, suggesting that PHB2-mediated regulation of mitophagy may serve as a novel strategy for downregulating the expression of platelet activation genes. Although further research into mitophagy is required, the present study suggested that PHB2 may serve as a novel therapeutic target for thrombosis-related diseases due to its unique localization on the mitochondrial membrane.

Green Fluorescent Terbium (III) Complex Doped Silica Nanoparticles.

The low photostability of conventional organic dyes and the toxicity of cadmium-based luminescent quantum dots have prompted the development of novel probes for in vitro and in vivo labelling. Here, a new fluorescent lanthanide probe based on silica nanoparticles is fabricated and investigated for optically traceable in vitro translocator protein (TSPO) targeting. The targeting and detection of TSPO receptor, overexpressed in several pathological states, including neurodegenerative diseases and cancers, may provide valuable information for the early diagnosis and therapy of human disorders. Green fluorescent terbium(III)-calix[4]arene derivative complexes are encapsulated within silica nanoparticles and surface functionalized amine groups are conjugated with selective TSPO ligands based on a 2-phenylimidazo[1,2-a]pyridine acetamide structure containing derivatizable carboxylic groups. The photophysical properties of the terbium complex, promising for biological labelling, are demonstrated to be successfully conveyed to the realized nanoarchitectures. In addition, the high degree of biocompatibility, assessed by cell viability assay and the selectivity towards TSPO mitochondrial membrane receptors, proven by subcellular fractional studies, highlight targeting potential of this nanostructure for in vitro labelling of mitochondria.

Neuroinflammatory Changes in Relation to Cerebrospinal Fluid Viral Load in Simian Immunodeficiency Virus Encephalitis.

The exact cause of neurocognitive dysfunction in HIV-positive patients despite successful control of the infection in the periphery is not completely understood. One suggested mechanism is a vicious cycle of microglial activation and release of proinflammatory chemokines/cytokines that eventually leads to neuronal loss and dysfunction. However, the exact role of microglial activation in the earliest stages of the infection with high cerebrospinal fluid (CSF) viral loads (VL) is unclear. In this study, we imaged the translocator protein (TSPO), a mitochondrial membrane receptor known to be upregulated in activated microglia and macrophages, in rhesus macaques before and multiple times after inoculation with a neurotropic simian immunodeficiency virus (SIV) strain (SIVsm804E), using 18F-DPA714 positron emission tomography (PET). The whole-brain standardized uptake values of TSPO at equilibrium reflecting total binding (SUVT) and binding potentials (BPND) were calculated and correlated with CSF and serum markers of disease, and a corresponding postmortem immunostaining analysis was also performed. SUVT was found to be inversely correlated with both CSF VL and monocyte chemoattractant protein 1 (MCP-1) levels. In SIV-infected macaques with very high CSF VL at necropsy (>106 copies/ml), we found decreased TSPO binding by PET, and this was supported by immunostaining which showed glial and neuronal apoptosis rather than microglial activation. On the other hand, with only moderately elevated CSF VL (∼104 copies/ml), we found increased TSPO binding as well as focal and diffuse microglial activation on immunostaining. Our results in the SIV-infected macaque model provide insights into the relationship between HIV neuropathology and CSF VL at various stages of the disease.IMPORTANCE Neurological and cognitive problems are a common complication of HIV infection and are prevalent even in treated individuals. Although the molecular processes underlying brain involvement with HIV are not completely understood, inflammation is suspected to play a significant role. Our work presents an in vivo assessment of neuroinflammation in an animal model of HIV, the simian immunodeficiency virus (SIV)-infected rhesus macaque. Using positron emission tomography (PET) imaging, we identified changes in brain inflammation after inoculation with SIV over time. Interestingly, we found decreased binding of the PET ligand in the presence of very high cerebrospinal fluid (CSF) viral loads. These findings were supported by immunostaining which showed marked glial loss instead of inflammation. This study provides insight into glial and neuronal changes associated with very high CSF viral load and could reflect similar changes occurring in HIV-infected patients.

Tracking Macrophage Infiltration in a Mouse Model of Pancreatic Cancer with the Positron Emission Tomography Tracer [11C]PBR28

BackgroundThe tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) contains abundant immunosuppressive tumor-associated macrophages. High level of infiltration is associated with poor outcome and is thought to represent a major roadblock to lymphocyte-based immunotherapy. Efforts to block macrophage infiltration have been met with some success, but noninvasive means to track tumor-associated macrophagess in PDAC are lacking. Translocator protein (TSPO) is a mitochondrial membrane receptor which is upregulated in activated macrophages. We sought to identify if a radiotracer-labeled cognate ligand could track macrophages in PDAC. Materials and MethodsA murine PDAC cell line was established from a transgenic mouse with pancreas-specific mutations in KRAS and p53. After confirming lack of endogenous TSPO expression, tumors were established in syngeneic mice. A radiolabeled TSPO-specific ligand ([11C] peripheral benzodiazepine receptor [PBR]28) was delivered intravenously, and tumor uptake was assessed by autoradiography, ex vivo, or micro-positron emission tomography imaging. ResultsResected tumors contained abundant macrophages as determined by immunohistochemistry and flow cytometry. Immunoblotting revealed murine macrophages expressed TSPO with increasing concentration on activation and polarization. Autoradiography of resected tumors confirmed [11C]PBR28 uptake, and whole mount sections demonstrated the ability to localize tumors. To confirm the findings were macrophage specific, experiments were repeated in CD11b-deficient mice, and the radiotracer uptake was diminished. Micro-positron emission tomography imaging validated radiotracer uptake and tumor localization in a clinically applicable manner. ConclusionsAs new immunotherapeutics reshape the PDAC microenvironment, tools are needed to better measure and track immune cell subsets. We have demonstrated the potential to measure changes in macrophage infiltration in PDAC using [11C]PBR28.

Adaptations Required for Mitochondrial Import following Mitochondrial to Nucleus Gene Transfer of Ribosomal Protein S10

The minimal requirements to support protein import into mitochondria were investigated in the context of the phenomenon of ongoing gene transfer from the mitochondrion to the nucleus in plants. Ribosomal protein 10 of the small subunit is encoded in the mitochondrion in soybean and many other angiosperms, whereas in several other species it is nuclear encoded and thus must be imported into the mitochondrial matrix to function. When encoded by the nuclear genome, it has adopted different strategies for mitochondrial targeting and import. In lettuce (Lactuca sativa) and carrot (Daucus carota), Rps10 independently gained different N-terminal extensions from other genes, following transfer to the nucleus. (The designation of Rps10 follows the following convention. The gene is indicated in italics. If encoded in the mitochondrion, it is rps10; if encoded in the nucleus, it is Rps10.) Here, we show that the N-terminal extensions of Rps10 in lettuce and carrot are both essential for mitochondrial import. In maize (Zea mays), Rps10 has not acquired an extension upon transfer but can be readily imported into mitochondria. Deletion analysis located the mitochondrial targeting region to the first 20 amino acids. Using site directed mutagenesis, we changed residues in the first 20 amino acids of the mitochondrial encoded soybean (Glycine max) rps10 to the corresponding amino acids in the nuclear encoded maize Rps10 until import was achieved. Changes were required that altered charge, hydrophobicity, predicted ability to form an amphipathic alpha-helix, and generation of a binding motif for the outer mitochondrial membrane receptor, translocase of the outer membrane 20. In addition to defining the changes required to achieve mitochondrial localization, the results demonstrate that even proteins that do not present barriers to import can require substantial changes to acquire a mitochondrial targeting signal.

Expression, Purification, and in Vitro Characterization of the Human Outer Mitochondrial Membrane Receptor Human Translocase of the Outer Mitochondrial Membrane 20

In order to investigate the biochemical properties of the mitochondrial outer membrane receptor, hTom20, involved in protein recognition, the cytosolic domain of this receptor was overexpressed and purified to homogeneity. A four-step purification including the purification of thrombin is described as well as an analysis of the function of the highly purified hTom20 protein. The receptor was concentrated and the subsequent aggregation behavior was investigated in order to understand the function of the single cysteine in the cytosolic domain as well as the function of the proposed “glutamine face” for the structure of the protein. It was found that specific dimerization of the cytosolic domain of hTom20 is necessary in order to prevent aggregation of the protein. In addition, the cysteine and the glutamine face are important for the stability of the protein. We propose that the function of the cysteine is to promote dimerization as found in the absence of dithiothreitol.

Evidence for a diazepam-binding inhibitor (DBI) benzodiazepine receptor-like mechanism in ecdysteroidogenesis by the insect prothoracic gland.

The diazepam-binding inhibitor (DBI) is a 10-kDa highly evolutionarily conserved multifunctional protein. In mammals, one of DBI's functions is in the activation of steroid hormone biosynthesis via binding to a specific outer mitochondrial membrane receptor (benzodiazepine receptor, BZD) and promoting cholesterol transport to the inner membrane. In this work, a multitiered approach was utilized to study the role of this receptor-like activity in ecdysteroidogenesis by larval insect prothoracic glands (PGs). First, both DBI protein and messenger RNA (mRNA) levels were correlated with peak PG ecdysteroid production. In vitro ecdysteroid production was stimulated by the diazepam analogue FGIN 1-27 and inhibited anti-DBI antibodies. The DBI protein was found distributed throughout PG cells, including regions of dense mitochondria, supposed subcellular sites of ecdysteroid synthesis. Finally, a potential mitochondrial BZD receptor in PG cells was demonstrated by photoaffinity labeling. These results suggest an important role for the insect DBI in the stimulation of steroidogenesis by prothoracic glands and indicate that a pathway for cholesterol mobilization leading to the production of steroid hormones appears to be conserved between arthropods and mammals.

Characterization of the N-terminal targeting signal binding domain of the mitochondrial outer membrane receptor, Tom20.

hTom20 is an outer mitochondrial membrane receptor involved in protein translocation. The cytosolic domain (aa30-145) and selected truncated versions of this domain were overexpressed and purified to study the structure-function relationship of this protein. Our studies reveal that the secondary structure of the cytosolic domain is very resistant to unfolding by guanidine-HCl and urea and is stabilized mainly by hydrophobic interactions. However, the tertiary structure of the N-terminal targeting signal binding domain (aa30-90) is more flexible. The first 30 amino acids of the cytosolic domain (aa30-60) are involved in recognizing N-terminal targeting signals and in stabilizing the cytosolic domain on the lipid surface. Moreover, we show that specifically aa30-48 interact with the membrane surface; a construct containing aa48-145 will only bind to the membrane surface in the presence of an N-terminal targeting signal peptide. The C-terminal region of hTom20 (aa141-145) interacts with the N-terminal region of hTom20, helping to stabilize the proper conformation of the N-terminal targeting signal binding domain. Finally, hTom20 interacts with the N-terminal targeting signal of preornithine carbamyl transferase fused to dihydrofolate reductase very weakly (Kd = 8 microM), as would be expected if this interaction was the first in a series orchestrated by the import receptor complex to draw the targeted protein into the mitochondrion.

The variant human isovaleryl-CoA dehydrogenase gene responsible for type II isovaleric acidemia determines an RNA splicing error, leading to the deletion of the entire second coding exon and the production of a truncated precursor protein that interacts poorly with mitochondrial import receptors.

Isovaleryl-CoA dehydrogenase (IVD) is a mitochondrial enzyme involved in leucine metabolism. Previous studies of fibroblasts from patients with isovaleric acidemia (IVA), an inherited defect in IVD, have revealed that IVD precursor protein produced by type II IVA cells is 3 kDa smaller than normal and is processed inefficiently to a mature form which is also 3 kDa smaller than normal. Using the polymerase chain reaction, we have identified a 90-base pair deletion encompassing bases 145-234 in type II IVD cDNA. This deletion is caused by an error in RNA splicing and predicts the in-frame deletion of 30 amino acids beginning with leucine 20 of the mature IVD. The rate of leader peptide cleavage by purified mitochondrial leader peptidases was similar for the variant and normal precursor IVDs expressed in vitro, and radiosequencing confirmed that both mature proteins contain identical amino termini. In vitro import studies showed that the efficiency of overall mitochondrial import of type II variant IVD precursor was approximately 30% of normal, as was its binding to the mitochondrial surface. Unlike its normal counterpart, the bound variant IVD precursor was readily released. These data suggest that binding of the variant protein to mitochondrial membrane receptors per se is hindered, resulting in the inefficient mitochondrial processing.

Hexokinase receptors: preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites.

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude "mitochondrial" fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude "mitochondrial fraction," a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to nonmitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.

Hexokinase-binding properties of the mitochondrial VDAC protein: Inhibition by DCCD and location of putative DCCD-binding sites

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [14C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257-265 and 275-283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.