PPIL4 is essential for brain angiogenesis and implicated in intracranial aneurysms in humans.

Intracranial aneurysm (IA) rupture leads to subarachnoid hemorrhage, a sudden-onset disease that often causes death or severe disability. Although genome-wide association studies have identified common genetic variants that increase IA risk moderately, the contribution of variants with large effect remains poorly defined. Using whole-exome sequencing, we identified significant enrichment of rare, deleterious mutations in PPIL4, encoding peptidyl-prolyl cis-trans isomerase-like 4, in both familial and index IA cases. Ppil4 depletion in vertebrate models causes intracerebral hemorrhage, defects in cerebrovascular morphology and impaired Wnt signaling. Wild-type, but not IA-mutant, PPIL4 potentiates Wnt signaling by binding JMJD6, a known angiogenesis regulator and Wnt activator. These findings identify a novel PPIL4-dependent Wnt signaling mechanism involved in brain-specific angiogenesis and maintenance of cerebrovascular integrity and implicate PPIL4 gene mutations in the pathogenesis of IA.

Identification of interacting proteins of Arabidopsis cyclophilin38 (AtCYP38) via multiple screening approaches reveals its possible broad functions in chloroplasts.

AtCYP38, a thylakoid lumen localized immunophilin, is found to be essential for photosystem II assembly and maintenance, but how AtCYP38 functions in chloroplast remains unknown. Based on previous functional studies and its crystal structure, we hypothesize that AtCYP38 should function via binding its targets or cofactors in the thylakoid lumen. To identify potential interacting proteins of AtCYP38, we first adopted ATTED-II and STRING web-tools, and found 12 proteins functionally related to AtCYP38. We then screened a yeast two-hybrid library including an Arabidopsis genome wide cDNA with different domain of AtCYP38, and five thylakoid lumen-localized targets were identified. In order to specifically search interacting proteins of AtCYP38 in the thylakoid lumen, we generated a yeast two-hybrid mini library including the thylakoid lumenal proteins and lumenal fractions of thylakoid membrane proteins, and we obtained six thylakoid membrane proteins and nine thylakoid lumenal proteins as interacting proteins of AtCYP38. The interactions between AtCYP38 and several potential targets were further confirmed via pull-down and co-immunoprecipitation assays. Together, a couple of new potential candidate interacting proteins of AtCYP38 were identified, and the results will lay a foundation for unveiling the regulatory mechanisms in photosynthesis by AtCYP38.

CYP20-3 deglutathionylates 2-CysPRX A and suppresses peroxide detoxification during heat stress.

In plants, growth-defense trade-offs occur because of limited resources, which demand prioritization towards either of them depending on various external and internal factors. However, very little is known about molecular mechanisms underlying their occurrence. Here, we describe that cyclophilin 20-3 (CYP20-3), a 12-oxo-phytodienoic acid (OPDA)-binding protein, crisscrosses stress responses with light-dependent electron reactions, which fine-tunes activities of key enzymes in plastid sulfur assimilations and photosynthesis. Under stressed states, OPDA, accumulates in the chloroplasts, binds and stimulates CYP20-3 to convey electrons towards serine acetyltransferase 1 (SAT1) and 2-Cys peroxiredoxin A (2CPA). The latter is a thiol-based peroxidase, protecting and optimizing photosynthesis by reducing its toxic byproducts (e.g., H2O2). Reduction of 2CPA then inactivates its peroxidase activity, suppressing the peroxide detoxification machinery, whereas the activation of SAT1 promotes thiol synthesis and builds up reduction capacity, which in turn triggers the retrograde regulation of defense gene expressions against abiotic stress. Thus, we conclude that CYP20-3 is a unique metabolic hub conveying resource allocations between plant growth and defense responses (trade-offs), ultimately balancing optimal growth phonotype.

Endomembrane Protein Trafficking Regulated by a TvCyP2 Cyclophilin in the Protozoan Parasite, Trichomonas vaginalis.

In Trichomonas vaginalis, the TvCyP1-catalyzed conformational switches of two glycinyl-prolyl imide bonds in Myb3 were previously shown to regulate the trafficking of Myb3 from cytoplasmic membrane compartments towards the nucleus. In this study, TvCyP2 was identified as a second cyclophilin that binds to Myb3 at the same dipeptide motifs. The enzymatic proficiency of TvCyP2, but not its binding to Myb3, was aborted by a mutation of Arg75 in the catalytic domain. TvCyP2 was localized to the endoplasmic reticulum with a weak signal that extensively extends into the cytoplasm as well as to the plasma membrane according to an immunofluorescence assay. Moreover, TvCyP2 was co-enriched with TvCyP1 and Myb3 in various membrane fractions purified by differential and gradient centrifugation. TvCyP2 was found to proficiently enzymatically regulate the distribution of TvCyP1 and Myb3 among purified membrane fractions, and to localize TvCyP1 in hydrogenosomes and on plasma membranes. Protein complexes immunoprecipitated from lysates of cells overexpressing TvCyP1 and TvCyP2 were found to share some common components, like TvCyP1, TvCyP2, TvBip, Myb3, TvHSP72, and the hydrogenosomal heat shock protein 70 (HSP70). Direct interaction between TvCyP1 and TvCyP2 was confirmed by a GST pull-down assay. Fusion of vesicles with hydrogenosomes was observed by transmission electron microscopy, whereas TvCyP1, TvCyP2, and Myb3 were each detected at the fusion junction by immunoelectron microscopy. These observations suggest that T. vaginalis may have evolved a novel protein trafficking pathway to deliver proteins among the endomembrane compartments, hydrogenosomes and plasma membranes.

ROS-related mitochondrial dysfunction in skeletal muscle of an ALS mouse model during the disease progression.

In amyotrophic lateral sclerosis (ALS), mitochondrial dysfunction and oxidative stress form a vicious cycle that promotes neurodegeneration and muscle wasting. To quantify the disease-stage-dependent changes of mitochondrial function and their relationship to the generation of reactive oxygen species (ROS), we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP transgenes, in which mt-cpYFP detects dynamic changes of ROS-related mitoflash events at individual mitochondria level. Compared with wild type mice, mitoflash activity in the SOD1G93A (G93A) mouse muscle showed an increased flashing frequency prior to the onset of ALS symptom (at the age of 2 months), whereas the onset of ALS symptoms (at the age of 4 months) is associated with drastic changes in the kinetics property of mitoflash signal with prolonged full duration at half maximum (FDHM). Elevated levels of cytosolic ROS in skeletal muscle derived from the SOD1G93A mice were confirmed with fluorescent probes, MitoSOX™ Red and ROS Brite™570. Immunoblotting analysis of subcellular mitochondrial fractionation of G93A muscle revealed an increased expression level of cyclophilin D (CypD), a regulatory component of the mitochondrial permeability transition pore (mPTP), at the age of 4 months but not at the age of 2 months. Transient overexpressing of SOD1G93A in skeletal muscle of wild type mice directly promoted mitochondrial ROS production with an enhanced mitoflash activity in the absence of motor neuron axonal withdrawal. Remarkably, the SOD1G93A-induced mitoflash activity was attenuated by the application of cyclosporine A (CsA), an inhibitor of CypD. Similar to the observation with the SOD1G93A transgenic mice, an increased expression level of CypD was also detected in skeletal muscle following transient overexpression of SOD1G93A. Overall, this study reveals a disease-stage-dependent change in mitochondrial function that is associated with CypD-dependent mPTP opening; and the ALS mutation SOD1G93A directly contributes to mitochondrial dysfunction in the absence of motor neuron axonal withdrawal.

Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift.

Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1-10 µm), whereas higher levels (0.5-1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 µm to 1 mm H2O2), with maximal effects at 500 µm H2O2 H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 µm H2O2 However, higher H2O2 levels (≥50 µm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation.

Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin.

In the course of thrombosis, platelets are exposed to a variety of activating stimuli classified as 'strong' (e.g. thrombin and collagen) or 'mild' (e.g. ADP). In response, activated platelets adhere to injured vasculature, aggregate, and stabilise the three-dimensional fibrin scaffold of the expanding thrombus. Since 'strong' stimuli also induce opening of the mitochondrial permeability transition pore (MPTP) in platelets, the MPTP-enhancer Cyclophilin D (CypD) has been suggested as a critical pharmacological target to influence thrombosis. However, it is poorly understood what role CypD plays in the platelet response to 'mild' stimuli which act independently of MPTP. Furthermore, it is unknown how CypD influences platelet-driven clot stabilisation against enzymatic breakdown (fibrinolysis). Here we show that treatment of human platelets with Cyclosporine A (a cyclophilin-inhibitor) boosts ADP-induced adhesion and aggregation, while genetic ablation of CypD in murine platelets enhances adhesion but not aggregation. We also report that platelets lacking CypD preserve their integrity in a fibrin environment, and lose their ability to render clots resistant against fibrinolysis. Our results indicate that CypD has opposing haemostatic roles depending on the stimulus and stage of platelet activation, warranting a careful design of any antithrombotic strategy targeting CypD.

Cyclophilin B Deficiency Causes Abnormal Dentin Collagen Matrix.

Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.

The Conserved RNA Binding Cyclophilin, Rct1, Regulates Small RNA Biogenesis and Splicing Independent of Heterochromatin Assembly.

RNAi factors and their catalytic activities are essential for heterochromatin assembly in S. pombe. This has led to the idea that siRNAs can promote H3K9 methylation by recruiting the cryptic loci regulator complex (CLRC), also known as recombination in K complex (RIKC), to the nucleation site. The conserved RNA-binding protein Rct1 (AtCyp59/SIG-7) interacts with splicing factors and RNA polymerase II. Here we show that Rct1 promotes processing of pericentromeric transcripts into siRNAs via the RNA recognition motif. Surprisingly, loss of siRNA in rct1 mutants has no effect on H3K9 di- or tri-methylation, resembling other splicing mutants, suggesting that post-transcriptional gene silencing per se is not required to maintain heterochromatin. Splicing of the Argonaute gene is also defective in rct1 mutants and contributes to loss of silencing but not to loss of siRNA. Our results suggest that Rct1 guides transcripts to the RNAi machinery by promoting splicing of elongating non-coding transcripts.

Vital role for cyclophilin B (CypB) in asexual development, dimorphic transition and virulence of Beauveria bassiana.

Cyclophilin B (CypB) was previously revealed as one of many putative secretory proteins in the transcriptome of Beauveria bassiana infection to a lepidopteran pest. Here we show a main localization of CypB in hyphal cell walls and septa and its essential role in the in vitro and in vivo asexual cycles of the fungal insect pathogen. Deletion of cypB reduced colony growth by 16-42% on two rich media and 30 scant media with different carbon or nitrogen sources. The deletion mutant suffered a delayed conidiation on a standard medium and a final 47% reduction in conidial yield, accompanied with drastic transcript depression of several key genes required for conidiation and conidial maturation. The mutant conidia required 10h longer to germinate 50% at optimal 25°C than wild-type conidia. Intriguingly, cultivation of the mutant conidia in a trehalose-peptone broth mimic to insect hemolymph resulted in 83% reduction in blastospore yield but only slight decrease in biomass level, indicating severe defects in transition of hyphae to blastospores. LT50 for the deletion mutant against Galleria mellonella larvae through normal cuticle infection was prolonged to 7.4d from a wild-type estimate of 4.7d. During colony growth, additionally, the deletion mutant displayed hypersensitivity to Congo red, menadione, H2O2 and heat shock but increased tolerance to cyclosporine A and rapamycin. All of changes were restored by targeted gene complementation. Altogether, CypB takes part in sustaining normal growth, aerial conidiation, conidial germination, dimorphic transition, stress tolerance and pathogenicity in B. bassiana.

The cyclophilin D/Drp1 axis regulates mitochondrial fission contributing to oxidative stress-induced mitochondrial dysfunctions in SH-SY5Y cells.

Oxidative stress plays a central role in the pathogenesis of various neurodegenerative diseases. Increasing evidences have demonstrated that structural abnormalities in mitochondria are involved in oxidative stress related nerve cell damage. And Drp1 plays a critical role in mitochondrial dynamic imbalance insulted by oxidative stress-derived mitochondria. However, the status of mitochondrial fusion and fission pathway and its relationship with mitochondrial properties such as mitochondrial membrane permeability transition pore (mPTP) have not been fully elucidated. Here, we demonstrated for the first time the role of Cyclophilin D (CypD), a crucial component for mPTP formation, in the regulation of mitochondrial dynamics in oxidative stress treated nerve cell. We observed that CypD-mediated phosphorylation of Drp1 and subsequently augmented Drp1 recruitment to mitochondria and shifts mitochondrial dynamics toward excessive fission, which contributes to the mitochondrial structural and functional dysfunctions in oxidative stress-treated nerve cells. CypD depletion or over expression accompanies mitochondrial dynamics/functions recovery or aggravation separately. We also demonstrated first time the link between the CypD to mitochondrial dynamics. Our data offer new insights into the mechanism of mitochondrial dynamics which contribute to the mitochondrial dysfunctions, specifically the role of CypD in Drp1-mediated mitochondrial fission. The protective effect of CsA, or other molecules affecting the function of CypD hold promise as a potential novel therapeutic strategy for governing oxidative stress pathology via mitochondrial pathways.

The Mitochondrial Permeability Transition Pore Regulator Cyclophilin D Exhibits Tissue-Specific Control of Metabolic Homeostasis.

The mitochondrial permeability transition pore (mPTP) is a key regulator of mitochondrial function that has been implicated in the pathogenesis of metabolic disease. Cyclophilin D (CypD) is a critical regulator that directly binds to mPTP constituents to facilitate the pore opening. We previously found that global CypD knockout mice (KO) are protected from diet-induced glucose intolerance; however, the tissue-specific function of CypD and mPTP, particularly in the control of glucose homeostasis, has not been ascertained. To this end, we performed calcium retention capacity (CRC) assay to compare the importance of CypD in the liver versus skeletal muscle. We found that liver mitochondria are more dependent on CypD for mPTP opening than skeletal muscle mitochondria. To ascertain the tissue-specific role of CypD in metabolic homeostasis, we generated liver-specific and muscle-specific CypD knockout mice (LKO and MKO, respectively) and fed them either a chow diet or 45% high-fat diet (HFD) for 14 weeks. MKO mice displayed similar body weight gain and glucose intolerance compared with wild type littermates (WT), whereas LKO mice developed greater visceral obesity, glucose intolerance and pyruvate intolerance compared with WT mice. These findings demonstrate that loss of muscle CypD is not sufficient to alter whole body glucose metabolism, while the loss of liver CypD exacerbates obesity and whole-body metabolic dysfunction in mice fed HFD.

Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria.

Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca2+ uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca2+-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca2+-induced membrane depolarisation and Ca2+ retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature.

The emerging role of calcium-modulating cyclophilin ligand in posttranslational insertion of tail-anchored proteins into the endoplasmic reticulum membrane.

Tail-anchored (TA) proteins, a class of membrane proteins having an N-terminal cytoplasmic region anchored to the membrane by a single C-terminal transmembrane domain, are posttranslationally inserted into the endoplasmic reticulum (ER) membrane. In yeasts, the posttranslational membrane insertion is mediated by the Guided Entry of TA Proteins (GET) complex. Get3, a cytosolic ATPase, targets newly synthesized TA proteins to the ER membrane, where Get2 and Get3 constitute the Get3 receptor driving the membrane insertion. While mammalian cells employ TRC40 and WRB, mammalian homologs of Get3 and Get1, respectively, they lack the gene homologous to Get2. We recently identified calcium-modulating cyclophilin ligand (CAML) as a TRC40 receptor, indicating that CAML was equivalent to Get2 in the context of the membrane insertion. On the other hand, CAML has been well characterized as a signaling molecule that regulates various biological processes, raising the question of how the two distinct actions of CAML, the membrane insertion and the signal transduction, are assembled. In this review, we summarize recent progress of the molecular mechanism of the membrane insertion of TA proteins and discuss the possibility that CAML could sense the various signals at the ER membrane, thereby controlling TA protein biogenesis.

Alisporivir inhibition of hepatocyte cyclophilins reduces HBV replication and hepatitis B surface antigen production.

BACKGROUND & AIMS: Cyclophilins are host factors required for hepatitis C virus replication. Cyclophilin inhibitors such as alisporivir have shown strong anti-hepatitis C virus activity in vitro and in clinical studies. However, little is known about whether hepatocyte cyclophilins are involved in the hepatitis B virus (HBV) life cycle. We investigated the effects of 2 cyclophilin inhibitors (alisporivir and NIM811) on HBV replication and hepatitis B surface antigen (HBsAg) production in cell lines. METHODS: Liver-derived cell lines producing full-length HBV and HBsAg particles, owing to stable (HepG2215) or transient (HuH-7) transfection, or infected with HBV (HepaRG cells; Invitrogen [Carlsbad, CA]), were incubated with alisporivir or NIM811 alone, or alisporivir in combination with a direct antiviral (telbivudine). The roles of individual cyclophilins in drug response was evaluated by small interfering RNA knockdown of cyclophilin (CYP)A, CYPC, or CYPD in HepG2215 cells, or CYPA knockdown in HuH-7 cells. The kinetics of antiviral activity were assessed based on levels of HBV DNA and HBsAg and Southern blot analysis. RESULTS: In HepG2215, HuH-7, and HepaRG cells, alisporivir reduced intracellular and secreted HBV DNA, in a dose-dependent manner. Knockdown of CYPA, CYPC, or CYPD (reduced by 80%) significantly reduced levels of HBV DNA and secreted HBsAg. Knockdown of CYPA significantly reduced secretion of HBsAg, leading to accumulation of intracellular HBsAg; the addition of alisporivir greatly reduced levels of HBsAg in these cells. The combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone. CONCLUSIONS: Alisporivir inhibition of cyclophilins in hepatocyte cell lines reduces replication of HBV DNA and HBsAg production and secretion. These effects are potentiated in combination with direct antiviral agents that target HBV-DNA polymerase.

Cyclophilin D modulates mitochondrial acetylome.

RATIONALE: Mice lacking cyclophilin D (CypD(-/-)), a mitochondrial chaperone protein, have altered cardiac metabolism. As acetylation has been shown to regulate metabolism, we tested whether changes in protein acetylation might play a role in these metabolic changes in CypD(-/-) hearts. OBJECTIVE: Our aim was to test the hypothesis that loss of CypD alters the cardiac mitochondrial acetylome. METHODS AND RESULTS: To identify changes in lysine-acetylated proteins and to map acetylation sites after ablation of CypD, we subjected tryptic digests of isolated cardiac mitochondria from wild-type and CypD(-/-) mice to immunoprecipitation using agarose beads coupled to antiacetyl lysine antibodies followed by mass spectrometry. We used label-free analysis for the relative quantification of the 875 common peptides that were acetylated in wild-type and CypD(-/-) samples and found 11 peptides (10 proteins) decreased and 96 peptides (48 proteins) increased in CypD(-/-) samples. We found increased acetylation of proteins in fatty acid oxidation and branched-chain amino acid metabolism. To evaluate whether this increase in acetylation might play a role in the inhibition of fatty acid oxidation that was previously reported in CypD(-/-) hearts, we measured the activity of l-3-hydroxyacyl-CoA dehydrogenase, which was acetylated in the CypD(-/-) hearts. Consistent with the hypothesis, l-3-hydroxyacyl-CoA dehydrogenase activity was inhibited by ≈50% compared with the wild-type mitochondria. CONCLUSIONS: These results implicate a role for CypD in modulating protein acetylation. Taken together, these results suggest that ablation of CypD leads to changes in the mitochondrial acetylome, which may contribute to altered mitochondrial metabolism in CypD(-/-) mice.

Depressing mitochondria-reticulum interactions protects cardiomyocytes from lethal hypoxia-reoxygenation injury.

BACKGROUND: Under physiological conditions, Ca(2+) transfer from the endoplasmic reticulum (ER) to mitochondria might occur at least in part at contact points between the 2 organelles and involves the VDAC1/Grp75/IP3R1 complex. Accumulation of Ca(2+) into the mitochondrial matrix may activate the mitochondrial chaperone cyclophilin D (CypD) and trigger permeability transition pore opening, whose role in ischemia/reperfusion injury is well recognized. We questioned here whether the transfer of Ca(2+) from ER to mitochondria might play a role in cardiomyocyte death after hypoxia-reoxygenation. METHODS AND RESULTS: We report that CypD interacts with the VDAC1/Grp75/IP3R1 complex in cardiomyocytes. Genetic or pharmacological inhibition of CypD in both H9c2 cardiomyoblasts and adult cardiomyocytes decreased the Ca(2+) transfer from ER to mitochondria through IP3R under normoxic conditions. During hypoxia-reoxygenation, the interaction between CypD and the IP3R1 Ca(2+) channeling complex increased concomitantly with mitochondrial Ca(2+) content. Inhibition of either CypD, IP3R1, or Grp75 decreased protein interaction within the complex, attenuated mitochondrial Ca(2+) overload, and protected cells from hypoxia-reoxygenation. Genetic or pharmacological inhibition of CypD provided a similar effect in adult mice cardiomyocytes. Disruption of ER-mitochondria interaction via the downregulation of Mfn2 similarly reduced the interaction between CypD and the IP3R1 complex and protected against hypoxia-reoxygenation injury. CONCLUSIONS: Our data (1) point to a new role of CypD at the ER-mitochondria interface and (2) suggest that decreasing ER-mitochondria interaction at reperfusion can protect cardiomyocytes against lethal reperfusion injury through the reduction of mitochondrial Ca(2+) overload via the CypD/VDAC1/Grp75/IP3R1 complex.

Mitochondrial modulation decreases the bortezomib-resistance in multiple myeloma cells.

Multiple myeloma (MM) is an incurable hematological malignancy that causes most patients to eventually relapse and die from their disease. The 20S proteasome inhibitor bortezomib has emerged as an effective drug for MM treatment; however, intrinsic and acquired resistance to bortezomib has already been observed in MM patients. We evaluated the involvement of mitochondria in resistance to bortezomib-induced cell death in two different MM cell lines (bortezomib-resistant KMS20 cells and bortezomib-sensitive KMS28BM cells). Indices of mitochondrial function, including membrane potential, oxygen consumption rate and adenosine-5'-triphosphate and mitochondrial Ca(2+) concentrations, were positively correlated with drug resistance of KMS cell lines. Mitochondrial genes including CYPD, SOD2 and MCU were differentially expressed in KMS cells. Thus, changes in the expression of these genes lead to changes in mitochondrial activity and in bortezomib susceptibility or resistance, and their combined effect contributes to differential sensitivity or resistance of MM cells to bortezomib. In support of this finding, coadministration of bortezomib and 2-methoxyestradiol, a SOD inhibitor, rendered KMS20 cells sensitive to apoptosis. Our results provide new insight into therapeutic modalities for MM patients. Studying mitochondrial activity and specific mitochondrial gene expression in fresh MM specimens might help predict resistance to proapoptotic chemotherapies and inform clinical decision-making.

Secreted trypanosome cyclophilin inactivates lytic insect defense peptides and induces parasite calcineurin activation and infectivity.

The mechanisms by which Trypanosoma cruzi survives antimicrobial peptides and differentiates during its transit through the gastrointestinal tract of the reduviid vector are unknown. We show that cyclophilin, a peptidyl-prolyl isomerase secreted from T. cruzi epimastigotes, binds to and neutralizes the reduviid antimicrobial peptide trialysin promoting parasite survival. This is dependent on a singular proline residue in trialysin and is inhibited by the cyclophilin inhibitor cyclosporine A. In addition, cyclophilin-trialysin complexes enhance the production of ATP and reductase responses of parasites, which are inhibited by both calcineurin-specific inhibitors cyclosporine A and FK506. Calcineurin phosphatase activity of cyclophilin-trialysin-treated parasites was higher than in controls and was inhibited by preincubation by either inhibitor. Parasites exposed to cyclophilin-trialysin have enhanced binding and invasion of host cells leading to higher infectivity. Leishmanial cyclophilin also mediates trialysin protection and metabolic stimulation by T. cruzi, indicating that extracellular cyclophilin may be critical to adaptation in other insect-borne protozoa. This work demonstrates that cyclophilin serves as molecular sensor leading to the evasion and adaptive metabolic response to insect defense peptides.