Hinge Protein Research Articles

Knockout of the Complex III subunit Uqcrh causes bioenergetic impairment and cardiac contractile dysfunction

Ubiquinol cytochrome c reductase hinge protein (UQCRH) is required for the electron transfer between cytochrome c1 and c of the mitochondrial cytochrome bc1 Complex (CIII). A two-exon deletion in the human UQCRH gene has recently been identified as the cause for a rare familial mitochondrial disorder. Deletion of the corresponding gene in the mouse (Uqcrh-KO) resulted in striking biochemical and clinical similarities including impairment of CIII, failure to thrive, elevated blood glucose levels, and early death. Here, we set out to test how global ablation of the murine Uqcrh affects cardiac morphology and contractility, and bioenergetics. Hearts from Uqcrh-KO mutant mice appeared macroscopically considerably smaller compared to wildtype littermate controls despite similar geometries as confirmed by transthoracic echocardiography (TTE). Relating TTE-assessed heart to body mass revealed the development of subtle cardiac enlargement, but histopathological analysis showed no excess collagen deposition. Nonetheless, Uqcrh-KO hearts developed pronounced contractile dysfunction. To assess mitochondrial functions, we used the high-resolution respirometer NextGen-O2k allowing measurement of mitochondrial respiratory capacity through the electron transfer system (ETS) simultaneously with the redox state of ETS-reactive coenzyme Q (Q), or production of reactive oxygen species (ROS). Compared to wildtype littermate controls, we found decreased mitochondrial respiratory capacity and more reduced Q in Uqcrh-KO, indicative for an impaired ETS. Yet, mitochondrial ROS production was not generally increased. Taken together, our data suggest that Uqcrh-KO leads to cardiac contractile dysfunction at 9 weeks of age, which is associated with impaired bioenergetics but not with mitochondrial ROS production.Graphical abstractGlobal ablation of the Uqcrh gene results in functional impairment of CIII associated with metabolic dysfunction and postnatal developmental arrest immediately after weaning from the mother. Uqcrh-KO mice show dramatically elevated blood glucose levels and decreased ability of isolated cardiac mitochondria to consume oxygen (O2). Impaired development (failure to thrive) after weaning manifests as a deficiency in the gain of body mass and growth of internal organ including the heart. The relative heart mass seemingly increases when organ mass calculated from transthoracic echocardiography (TTE) is normalized to body mass. Notably, the heart shows no signs of collagen deposition, yet does develop a contractile dysfunction reflected by a decrease in ejection fraction and fractional shortening.

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Reperfusion mediates heme impairment with increased protein cysteine sulfonation of mitochondrial complex III in the post-ischemic heart

A serious consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which causes mitochondrial dysfunction. The cascading ROS can propagate and potentially induce heme bleaching and protein cysteine sulfonation (PrSO3H) of the mitochondrial electron transport chain. Herein we studied the mechanism of I/R-mediated irreversible oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the catalytic activity of complex III was significantly impaired. Spectroscopic analysis indicated that I/R mediated the destruction of hemes b and c + c1 in the mitochondria, supporting I/R-mediated complex III impairment. However, no significant impairment of complex III activity and heme damage were observed in mitochondria from the risk region of rat hearts subjected only to 30-min ischemia, despite a decreased state 3 respiration. In the I/R mitochondria, carbamidomethylated C122/C125 of cytochrome c1 via alkylating complex III with a down regulation of HCCS was exclusively detected, supporting I/R-mediated thioether defect of heme c1. LC-MS/MS analysis showed that I/R mitochondria had intensely increased complex III PrSO3H levels at the C236 ligand of the [2Fe2S] cluster of the Rieske iron‑sulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSO3H of the hinge protein at C65 and of cytochrome c1 at C140 and C220, which are confined in the intermembrane space. MS analysis also showed that I/R extensively enhanced the PrSO3H of the core 1 (uqcrc1) and core 2 (uqcrc2) subunits in the matrix compartment, thus supporting the conclusion that complex III releases ROS to both sides of the inner membrane during reperfusion. Analysis of ischemic mitochondria indicated a modest reduction from the basal level of complex III PrSO3H detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSO3H. In conclusion, reperfusion-mediated heme damage with increased PrSO3H controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart.

Genome-wide analysis of DNA methylation identifies the apoptosis-related gene UQCRH as a tumor suppressor in renal cancer.

DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5‐aza‐deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome‐wide using methylation array in reference to RNA‐sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor‐suppressive role of UQCRH. Moreover, 5‐aza‐deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B‐induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC.

Abstract 2091: DNA methylation enhances apoptosis resistance of renal cancer cells

Abstract Epigenetic modifications are important not only for carcinogenesis but also for metastasis of clear cell renal cell carcinoma (ccRCC). We previously reported that inflammation-related signaling is constitutively activated in advanced ccRCC through the formation of super enhancer. In addition to histone modification, methylation of the primary structure of DNA is implicated in the progression of ccRCC. Recent studies revealed that DNA hypermethylation was frequently observed in ccRCC and correlated with poor prognosis. However, unlike in other cancers, methylated genes responsible for cancer progression is still unclear in ccRCC. In this study, we tried to identify a target(s) for DNA methyltransferases (DNMTs) in ccRCC cells based on the genome-wide analysis. Its function for the regulation of cellular survival was examined. Our preclinical model of ccRCC using the serial orthotopic inoculation model exhibited the up-regulation of DNMT3B in advanced ccRCC. Pre-treatment of advanced ccRCC with 5-aza-dC attenuated a formation of a primary tumor through the induction apoptosis. DNA methylated sites were analyzed using methylation array in reference to RNA-sequencing data. As a result, ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissue was lower than normal adjacent tissue. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo. Our results imply that DNA methylation induces the decreased expression of potential tumor suppressor gene UQCRH, which is essential for the completion of apoptotic process of ccRCC cells. These findings may contribute to the understanding of molecular mechanisms involved in the drug resistance of ccRCC cells and provide an important insight for the improvement of the efficacy of existing medicines. Citation Format: Kosuke Miyakuni, Jun Nishida, Shogo Ehata, Kohei Miyazono. DNA methylation enhances apoptosis resistance of renal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2091.

UQCRH downregulation promotes Warburg effect in renal cell carcinoma cells

Ubiquinol-cytochrome c reductase hinge protein (UQCRH) is the hinge protein for the multi-subunit complex III of the mitochondrial electron transport chain and is involved in the electron transfer reaction between cytochrome c1 and c. Recent genome-wide transcriptomic and epigenomic profiling of clear cell renal cell carcinoma (ccRCC) by The Cancer Genome Atlas (TCGA) identified UQCRH as the top-ranked gene showing inverse correlation between DNA hypermethylation and mRNA downregulation. The function and underlying mechanism of UQCRH in the Warburg effect metabolism of ccRCC have not been characterized. Here, we verified the clinical association of low UQCRH expression and shorter survival of ccRCC patients through in silico analysis and identified KMRC2 as a highly relevant ccRCC cell line that displays hypermethylation-induced UQCRH extinction. Ectopic overexpression of UQCRH in KMRC2 restored mitochondrial membrane potential, increased oxygen consumption, and attenuated the Warburg effect at the cellular level. UQCRH overexpression in KMRC2 induced higher apoptosis and slowed down in vitro and in vivo tumor growth. UQCRH knockout by CRISPR/Cas9 had little impact on the metabolism and proliferation of 786O ccRCC cell line, suggesting the dispensable role of UQCRH in cells that have entered a Warburg-like state through other mechanisms. Together, our study suggests that loss of UQCRH expression by hypermethylation may promote kidney carcinogenesis through exacerbating the functional decline of mitochondria thus reinforcing the Warburg effect.

Abstract 4795: Metabolic vulnerability by UQCRH methylation in clear cell renal cell carcinoma

Abstract Background: UQCRH, subunit 6 of complex III in the electron transport chain of the mitochondria, is a hinge protein that stabilizes cytochrome c-c1 complex formation. UQCRH is the top-ranked gene by inverse correlation between DNA methylation and gene expression in clear cell renal carcinoma (ccRCC) in the Cancer Genome Atlas and Broad Institute Cancer Cell Line Encyclopedia databases. In the clinic, ccRCC patients with low UQCRH expression exhibit higher tumor stage and grade. While its downregulation is correlated with worse tumor prognosis, little is known about the mechanism through which UQCRH is involved in cancer progression. We propose that hypermethylation-induced downregulation of UQCRH inhibits complex III activity, thus lowering the efficiency of oxidative phosphorylation and shifting the metabolic balance of the cell toward aerobic glycolysis, which contributes to tumor proliferation. Methods: Two ccRCC cell lines were used, KMRC-2 and 786-O. We measured UQCRH and mRNA expression levels and identified which cell lines exhibited UQCRH downregulation. We used the global demethylation agent Decitabine (Dacogen) to test whether methylation of the UQCRH gene was present in the low-UQCRH cell line. UQCRH overexpression and knockout sublines were created for high and low-UQCRH cell lines, respectively. We characterized the metabolic preferences of the sublines and original cell lines and measured tumor proliferation in vitro and in vivo. We also studied the effect of ketogenic diet on cell proliferation in vitro. Results: While 786-O expressed normal levels of UQCRH, KMRC-2 expressed low levels of UQCRH, but its UQCRH expression was rescued by increasing concentrations of Decitabine. Sublines with low UQCRH, whether by hypermethylation or knockout, had higher extracellular acidification rate compared to their high-UQCRH counterparts. Although no significant difference was detected in cell proliferation in vitro, low-UQCRH tumors grew faster in vivo. Ketogenic diet experiments indicated that high UQCRH expression was positively correlated with more efficient beta-hydroxybutyrate metabolism and cellular proliferation. Beta-hydroxybutyrate was toxic to low-UQCRH sublines. Conclusion: Hypermethylation induced UQCRH downregulation in KMRC-2. Cells with lower UQCRH levels exhibited weaker mitochondrial membrane potential and were more reliant on aerobic glycolysis. While this metabolic reprogramming did not affect proliferation in vitro, low-UQCRH tumors grew faster in vivo. The metabolic shift also represents a vulnerability that could be targeted in the development of therapies to hinder tumor progression. The ketogenic diet showed promise as such a strategy. Citation Format: Yanting Luo, Louise Medina Bengtsson, Xin Lu. Metabolic vulnerability by UQCRH methylation in clear cell renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4795.

Increased cysteine sulfonation of Complex III with destruction of heme c mediates cardiac reperfusion injury via impairing catalytic function and enhancing FADH2‐linked superoxide production

One consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which can cause mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction results in impaired state 3 respiration and overproduction of superoxide, which can potentially induce protein cysteine sulfonation (PrSO3H) in mitochondria. Herein we employed UV/VIS spectroscopy and LC-MS/MS analysis to study the mechanism of I/R-mediated oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the activity of complex III was decreased by 31.0±4.1 % (n=7, p<0.001). Visible spectroscopic and immunoblotting analysis indicated that I/R mediated destruction of heme c (c-type heme) without affecting protein level of cytochrome c (cyt c) in the mitochondria. I/R thus mediates dysfunction of cyt c via heme destruction. However, no significant impairment of complex III activity and heme c destruction was observed in mitochondria isolated from the risk region of rat heart subjected to 30 min ischemia (no reperfusion) in vivo despite a decreased state 3 respiration. LC-MS/MS analysis showed enhanced formation of PrSO3H, which was consistent with mitochondrial redox state following I/R (EPR measurement of cyclic hydroxylamine oxidation). Mitochondria from I/R regions had intensely increased oxidative modification with sulfonation of cysteine (at C236) of 2Fe-2S cluster of Rieske iron-sulfur protein (uqcrfs1), which would impair the main electron transfer pathway controlled by the Q-cycle mechanism of complex III. LC-MS/MS analysis also indicated that I/R intensely increased complex III PrSO3H at the subunits of hinge protein, cytochrome c1, core 1 and core 2. Carbamidomethylated C122/C125 of the cytochrome c1 via alkylating complex III was exclusively detected in the post-ischemic mitochondria by LC-MS/MS, indicating I/R mediates cleavage of thioether bonds between heme c1 and apocytochrome c1. FADH2-linked ·O2− generation by mitochondrial SCR (supercomplex hosting complex II and complex III) was induced by succinate and evaluated by EPR spin trapping with DEPMPO. FADH2-linked ·O2− generation by normal mitochondrial SCR was minimized in the presence of a functional cyt c. Whereas, FADH2-linked ·O2− generation by the post-ischemic mitochondrial SCR was intensely increased by 123% in the presence of heme-depleted cyt c, and further enhanced by 227% under the conditions of increasing pO2 in vitro (from 0.23 mM O2 to 0.8 mM O2), suggesting that physiologic conditions of hyperoxygenation during reperfusion with heme-depleted cyt c mediated overproduction of FADH2-linked ·O2− by the post-ischemic mitochondria. In conclusion, reperfusion-induced PrSO3H with heme depletion of cyt c controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction via overproducing ·O2− in the post-ischemic heart. Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may necessitate lessening of PrSO3H and preservation of functional integrity of heme groups in mitochondria. Support or Funding Information NIH RO1HL083237 Enhancement of FADH2-linked superoxide generation by post-ischemic mitochondrial SCR (C vs B) in the presence of post-ischemic cyt c (D vs B) and hyperoxygenation (E vs D). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ITRAQ-based proteomic analysis of human umbilical vein endothelial cells with platelet endothelial aggregation receptor-1 knockdown.

The disorders of hemostasis and coagulation were believed to be the main contributors to the pathogenesis of pulmonary thromboembolism (PTE), and platelets are the basic factors regulating hemostasis and coagulation and play important roles in the process of thrombosis. This study investigated the proteome of human umbilical vein endothelial cells (HUVECs) with platelet endothelial aggregation receptor-1 (PEAR1) knockdown using the isobaric tags for relative and absolute quantitation (iTRAQ) method and analyzed the role of differential abundance proteins (DAPs) in the regulation of platelets aggregation. Our results showed that the conditioned media-culturing HUVECs with PEAR1 knockdown partially suppressed the adenosine diphosphate (ADP)-induced platelet aggregation. The proteomics analysis was performed by using the iTRAQ technique, and a total of 215 DAPs (124 protein was upregulated and 91 protein were downregulated) were identified. The Gene Ontology (GO) enrichment analysis showed that proteins related to platelet α granule, adenosine triphosphate metabolic process, and endocytosis were significantly enriched. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also identified the significant enrichment of endocytosis-related pathways. The real-time polymerase chain reaction assay confirmed that the expression of P2Y12 , mitochondrial carrier 2, NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, and ubiquinol-cytochrome c reductase hinge protein are significantly downregulated in the HUVECs with PEAR1 knockdown. In conclusion, our in vitro results implicated that DAPs induced by PEAR1 knockdown might contribute to the platelet aggregation. Proteomic studies by employing GO enrichment and KEGG pathway analysis suggested that the potential effects of DAPs on platelet aggregation may be linked to the balance of ADP synthesis or degradation in mitochondria.

Increased Protein Cysteine Sulfonation with Heme Destruction of Mitochondrial Complex III Mediates Cardiac Reperfusion Injury

A serious consequence of myocardial ischemia‐reperfusion injury (IR) is oxidative damage, which causes mitochondrial dysfunction. Such IR induced mitochondrial dysfunction results in impaired state 3 respiration and overproduction of superoxide. The cascading reactive oxygen species can propagate and potentially induce protein cysteine sulfonation (PrCSO3H) of mitochondrial proteins. Herein we employed UV/VIS spectroscopy and LC‐MS/MS analysis to study the mechanism of IR‐mediated oxidative injury of complex III in mitochondria from rat hearts subjected to 30 min of ischemia (coronary ligation) and 24 h of reperfusion in vivo. In the IR region, the activity of complex III was decreased by 31.0±4.1 % (n=7, p&lt;0.001). Visible spectroscopic analysis indicated that IR mediated destruction of heme b and hemes (c+c1) in the mitochondria, thus supporting IR‐induced impairment of the enzymatic activity of complex III. However, no significant impairment of complex III activity and heme destruction was observed in mitochondria isolated from the risk region of rat heart subjected to 30 min coronary ligation in vivo despite a decreased state 3 respiration detected in the ischemic mitochondria. LC‐MS/MS analysis showed formation of PrCSO3H, which was consistent with a redox analysis of mitochondria following IR (EPR measurement of oxidation of cyclic hydroxylamine). Mitochondria from IR regions had intensely increased oxidative modification with S‐sulfonation level on the cysteine ligand (at C236) of 2Fe‐2S cluster of Rieske iron‐sulfur protein (uqcrfs1), thus impairing the main electron transfer pathway controlled by the Q‐cycle mechanism of complex III. LC‐MS/MS analysis also indicated that IR‐mediated increased PrCSO3H of hinge protein at C65 (subunit 8, uqcrh) and cytochrome c1 subunit at C140 and C220, which weakened the interaction of hinge protein with cytochrome c1 and proper complex formation between cytochrome c1 and cytochrome c. LC‐MS/MS analysis further identified 4‐HNE (4‐hydroxylnonenal) adduct of cytochrome c1 at C140, suggesting involvement of IR‐induced lipid peroxidation in heme destruction and PrCSO3H of complex III. LC‐MS/MS analysis also showed that IR‐enhancing PrCSO3H of core 1 subunit (uqcrc1 at C69, C268, C380, C410, C445, C453) and core 2 subunit (uqcrc2 at C191), which may affect the binding of subunit 9 inhibitory peptide to core 1 and core 2 subunits and reactivate the MPP (matrix processing peptidase) activity of the core 1/core 2 subunits of mammalian complex III. LC‐MS/MS analysis of ischemic mitochondria has indicated a modest reduction from the basal level of complex III PrCSO3H detected in the mitochondria of sham control heart, suggesting that physiologic conditions of hypoxia during ischemia suppressed PrCSO3H, and physiologic conditions of hyperoxygenation during reperfusion mediated enhancement of complex III PrCSO3H. In conclusion, reperfusion‐mediated heme destruction with increased PrCSO3H controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction in the post‐ischemic heart. Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may related to the preservation of functional integrity of heme groups and protein structure within mitochondrial complex III.Support or Funding InformationNHLBI/NIH, HL083237This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The mitochondrial hinge protein, UQCRH, is a novel prognostic factor for hepatocellular carcinoma.

Alterations in mitochondrial respiration contribute to the development and progression of cancer via abnormal biogenesis, including generation of reactive oxygen species. Ubiquinol–cytochrome c reductase hinge protein (UQCRH) consists of the cytochrome bc1 complex serving respiration in mitochondria. In the present study, we analyzed UQCRH abnormalities in hepatocellular carcinoma (HCC) and its association with clinical outcomes of patients. UQCRH expression in HCC was determined via semiquantitative and quantitative real‐time reverse transcriptase polymerase chain reaction of 96 surgically resected HCC tissues positive for hepatitis B virus surface antigen. UQCRH was frequently overexpressed in HCC tissues (46.8%, based on 2.1‐fold cutoff). UQCRH overexpression was observed in HCCs with larger tumor size, poorer differentiation, or vascular invasion. Kaplan–Meier analysis revealed significantly shorter overall (P = 0.005) and recurrence‐free survival (P = 0.027) in patients with tumors overexpressing UQCRH. The prognostic impact of UQCRH was significant in subgroups of patients divided according to the α‐fetoprotein (AFP) level. The patient subgroup with higher AFP levels (≥20 ng/mL) exhibited significant differences in 5‐year overall (18.5% vs. 67.9%) and recurrence‐free survival rates (11.1% vs. 46.4%) between groups with and without UQCRH overexpression. In contrast, no marked survival differences were observed between subgroups with lower AFP levels (<20 ng/mL). Multivariate analysis defined UQCRH as an independent poor prognostic factor. Conclusively, our results indicate that UQCRH overexpression is correlated with poor outcomes of HCC patients. Furthermore, in patients grouped as high risk based on elevated AFP, lack of UQCRH overexpression could be a useful indicator for clinical treatment.

Identification of key genes in Gram‑positive and Gram‑negative sepsis using stochastic perturbation.

Sepsis is an inflammatory response to pathogens (such as Gram-positive and Gram-negative bacteria), which has high morbidity and mortality in critically ill patients. The present study aimed to identify the key genes in Gram-positive and Gram-negative sepsis. GSE6535 was downloaded from Gene Expression Omnibus, containing 17 control samples, 18 Gram-positive samples and 25 Gram-negative samples. Subsequently, the limma package in R was used to screen the differentially expressed genes (DEGs). Hierarchical clustering was conducted for the specific DEGs in Gram-negative and Gram-negative samples using cluster software and the TreeView software. To analyze the correlation of samples at the gene level, a similarity network was constructed using Cytoscape software. Functional and pathway enrichment analyses were conducted for the DEGs using DAVID. Finally, stochastic perturbation was used to determine the significantly differential functions between Gram-positive and Gram-negative samples. A total of 340 and 485 DEGs were obtained in Gram-positive and Gram-negative samples, respectively. Hierarchical clustering revealed that there were significant differences between control and sepsis samples. In Gram-positive and Gram-negative samples, myeloid cell leukemia sequence 1 was associated with apoptosis and programmed cell death. Additionally, NADH:ubiquinone oxidoreductase subunit S4 was associated with mitochondrial respiratory chain complex I assembly. Stochastic perturbation analysis revealed that NADH:ubiquinone oxidoreductase subunit B2 (NDUFB2), NDUFB8 and ubiquinol-cytochrome c reductase hinge protein (UQCRH) were associated with cellular respiration in Gram-negative samples, whereas large tumor suppressor kinase 2 (LATS2) was associated with G1/S transition of the mitotic cell cycle in Gram-positive samples. NDUFB2, NDUFB8 and UQCRH may be biomarkers for Gram-negative sepsis, whereas LATS2 may be a biomarker for Gram-positive sepsis. These findings may promote the therapies of sepsis caused by Gram-positive and Gram-negative bacteria.

In Vitro Evidence Supports Membrane Alanyl Aminopeptidase N as a Receptor for a Plant Virus in the Pea Aphid Vector.

Insect-borne plant viruses cause significant agricultural losses and jeopardize sustainable global food production. Although blocking plant virus transmission would allow for crop protection, virus receptors in insect vectors are unknown. Here we identify membrane alanyl aminopeptidase N (APN) as a receptor for pea enation mosaic virus (PEMV) coat protein (CP) in the gut of the pea aphid, Acyrthosiphon pisum, using a far-Western blot method. Pulldown and immunofluorescence binding assays and surface plasmon resonance were used to confirm and characterize CP-APN interaction. PEMV virions and a peptide comprised of PEMV CP fused to a proline-rich hinge (-P-) and green fluorescent protein (CP-P-GFP) specifically bound to APN. Recombinant APN expressed in Sf9 cells resulted in internalization of CP-P-GFP, which was visualized by confocal microscopy; such internalization is an expected hallmark of a functional gut receptor. Finally, in assays with aphid gut-derived brush border membrane vesicles, binding of CP-P-GFP competed with binding of GBP3.1, a peptide previously demonstrated to bind to APN in the aphid gut and to impede PEMV uptake into the hemocoel; this finding supports the hypothesis that GBP3.1 and PEMV bind to and compete for the same APN receptor. These in vitro data combined with previously published in vivo experiments (S. Liu, S. Sivakumar, W. O. Sparks, W. A. Miller, and B. C. Bonning, Virology 401:107-116, 2010, http://dx.doi.org/10.1016/j.virol.2010.02.009) support the identification of APN as the first receptor in a plant virus vector. Knowledge of this receptor will provide for technologies based on PEMV-APN interaction designed to block plant virus transmission and to suppress aphid populations. A significant proportion of global food production is lost to insect pests. Aphids, in addition to weakening plants by feeding on their sap, are responsible for transmitting about half of the plant viruses vectored by insects. Growers rely heavily on the application of chemical insecticides to manage both aphids and aphid-vectored plant viral disease. To increase our understanding of plant virus-aphid vector interaction, we provide in vitro evidence supporting earlier in vivo work for identification of a receptor protein in the aphid gut called aminopeptidase N, which is responsible for entry of the plant virus pea enation mosaic virus into the pea aphid vector. Enrichment of proteins found on the surface of the aphid gut epithelium resulted in identification of this first aphid gut receptor for a plant virus. This discovery is particularly important since the disruption of plant virus binding to such a receptor may enable the development of a nonchemical strategy for controlling aphid-vectored plant viruses to maximize food production.

The Ability to Form Homodimers Is Essential for RDM1 to Function in RNA-Directed DNA Methylation

RDM1 (RNA-DIRECTED DNA METHYLATION1) is a small plant-specific protein required for RNA-directed DNA methylation (RdDM). RDM1 interacts with RNA polymerase II (Pol II), ARGONAUTE4 (AGO4), and the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) and binds to methylated single stranded DNA. As the only protein identified so far that interacts directly with DRM2, RDM1 plays a pivotal role in the RdDM mechanism by linking the de novo DNA methyltransferase activity to AGO4, which binds short interfering RNAs (siRNAs) that presumably base-pair with Pol II or Pol V scaffold transcripts synthesized at target loci. RDM1 also acts together with the chromatin remodeler DEFECTIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1) and the structural-maintenance-of-chromosomes solo hinge protein DEFECTIVE IN MERISTEM SILENCING3 (DMS3) to form the DDR complex, which facilitates synthesis of Pol V scaffold transcripts. The manner in which RDM1 acts in both the DDR complex and as a factor bridging DRM2 and AGO4 remains unclear. RDM1 contains no known protein domains but a prior structural analysis suggested distinct regions that create a hydrophobic pocket and promote homodimer formation, respectively. We have tested several mutated forms of RDM1 altered in the predicted pocket and dimerization regions for their ability to complement defects in RdDM and transcriptional gene silencing, support synthesis of Pol V transcripts, form homodimers, and interact with DMS3. Our results indicate that the ability to form homodimers is essential for RDM1 to function fully in the RdDM pathway and may be particularly important during the de novo methylation step.

Molecular characterization and tissue expression profile of three novel ovine genes: ATP5O, NDUFA12 and UQCRH from muscle full-length cDNA library of black-boned sheep

Three novel ovine genes were obtained from muscle full-length cDNA library of black-boned sheep. Sequence analysis revealed that nucleotide sequences of these genes were not homologous to any of the known sheep or goat genes, but these genes have high similarity to ATP synthase subunit O (ATP5O), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 (NDUFA12) and ubiquinol-cytochrome c reductase hinge protein (UQCRH) genes of other mammal animals (accession number: FJ546085, FJ546078 and FJ546083). The alignment analysis showed that the ovine ATP5O, NDUFA12 and UQCRH genes and proteins have closer genetic relationships with the ATP5O, NDUFA12 and UQCRH genes and proteins from cattle. Conserved domain prediction showed that these three genes included OSCP, NDUFA12 superfamily and UCR-hinge superfamily domains respectively. The deduced sequence of ATP5O, NDUFA12 and UQCRH protein had 213, 145 and 91 amino acid residues, with a molecular weight of approximately 23419.66, 17089.50 and 10657.75Da and a theoretical isoelectric point of 9.90, 9.68 and 4.45. The secondary structure prediction revealed that 60% helix structure in ATP5O, 60% coils in NDUFA12 and no strand in UQCRH. One potential signal peptide structure in ATP5O protein were found. NDUFA12 and UQCRH have the extremely low possibility of signal peptides. Meanwhile, RasMol was used for visualizing the PDB files generated by Swiss-Model in cartoon or three-dimensional format. ATP5O and UQCRH protein were modeled by Swiss-Model. Tissue expression profile indicated that the ovine ATP5O, NDUFA12 and UQCRH genes could be expressed in all detected tissues including muscles, heart, liver, spleen, lung, kidney and adipose tissues, but the expression abundance of these genes were various in the different tissues. Our experiment supplied the primary foundation for further researches on these three ovine genes.

Impaired OXPHOS Complex III in Breast Cancer

We measured the mitochondrial oxidative phosphorylation (mtOXPHOS) activities of all five complexes and determined the activity and gene expression in detail of the Complex III subunits in human breast cancer cell lines and primary tumors. Our analysis revealed dramatic differences in activity of complex III between normal and aggressive metastatic breast cancer cell lines. Determination of Complex III subunit gene expression identified over expression and co-regulation of UQCRFS1 (encoding RISP protein) and UQCRH (encoding Hinge protein) in 6 out of 9 human breast tumors. Analyses of UQCRFS1/RISP expression in additional matched normal and breast tumors demonstrated an over expression in 14 out of 40 (35%) breast tumors. UQCRFS1/RISP knockdown in breast tumor cell line led to decreased mitochondrial membrane potential as well as a decrease in matrigel invasion. Furthermore, reduced matrigel invasion was mediated by reduced ROS levels coinciding with decreased expression of NADPH oxidase 2, 3, 4 and 5 involved in ROS production. These studies provide direct evidence for contribution of impaired mtOXPHOS Complex III to breast tumorigenesis.

Generating Global Protein Dynamics with Structure-From-FRET

The remarkable specificity and diversity of protein function are consequences of native conformational fluctuations, and more specifically transitions between conformational substates. Experimentally, these dynamics are increasingly resolvable (with high temporal detail) using single molecule Forster Resonance Energy Transfer (smFRET) methods, which can capture rare structural extensions not discernible with bulk methods. Still, even with multiple fluorophore-pairs, dye-separation trajectories lack the atomistic detail of crystallographic or NMR-based methods, and, at present, merging smFRET data with high-resolution structural data remains problematic. Moreover, many functionally-relevant transitions proceed within timescales far beyond those accessible to current molecular dynamics approaches. We address this common tradeoff between detail and sampling by posing the following inference problem: given a single dynamic distance constraint, what is the most likely protein structure? For a solution, our Kalman-filter-inspired approach, Structure-from-FRET, generates an atomistic protein trajectory most likely responsible for the observed smFRET distance constraint: a statistical inference inverse problem. As a general analysis and visualization tool, we apply the method to the model hinge protein adenylate kinase. Millisecond-resolution FRET efficiencies indicate approximate Lid/Core distance traces, which constrain inference of global protein geometry. Resultant trajectories span extended temporal (seconds) and conformational (tens of Angstroms) scales, without imposed potentials or perturbations between reference states. Here, the inter-residue distances constrain a state-space estimation with backbone atom coordinates as variables. Experimental (constraint) uncertainty is explicitly considered and each update step maximizes spatial information from previous ‘frames’. We compare generated trajectories against those from interpolative or elastic network approaches, but also with known PDB structures and long run MD simulations. Results suggest Structure-from-FRET is reliable for modeling novel biocatalysts, inhibitor targets, or other proteins for which multiple structures or atomistic simulations are unavailable.

UQCRH gene encoding mitochondrial Hinge protein is interrupted by a translocation in a soft-tissue sarcoma and epigenetically inactivated in some cancer cell lines.

We previously reported the identification of a novel zinc-finger gene, designated ZSG, fused to Ewing sarcoma gene (EWS) by a submicroscopic paracentric inversion of 22q12 in a small round cell sarcoma presenting a translocation t(1;22)(p34;q12). We report here the molecular cloning and characterization of the breakpoint in 1p34, which encompasses the gene coding for mitochondrial Hinge protein ubiquinol-cytochrome C reductase hinge gene (UQCRH). All the three breakpoints, two on 22q12 and one in 1p34, interrupt different genes: EWS, ZSG and UQCRH. We determined the genomic structure of UQCRH, characterized its splicing variants and identified a transcribed processed pseudogene. The analysis of UQCRH expression in normal tissues and cancer cell lines revealed absent expression of UQCRH in two ovarian and one breast cancer cell lines and reduced expression in a further breast carcinoma cell line. CpG island methylation upstream exon 1 was detected in all the three cell lines with absent expression. Moreover, treatment with demethylating agent 5-azacytidine restored UQCRH expression in OAW42 ovarian cancer cells. These data provide preliminary evidence of the inactivation of UQCRH gene in cancer either by structural rearrangements or epigenetic mechanisms.

Overexpressed Mitochondrial Hinge Protein, a Cytochromec-Binding Protein, Accelerates Apoptosis by Enhancing the Release of Cytochromecfrom Mitochondria

Holocytochromecreleased from mitochondria has been revealed to be one of the contributors of apoptosis. To investigate how the cytochromecprotein is released from mitochondria, we examined the effects of overexpression of the hinge protein, a cytochromec-binding protein, or cytochromecon apoptosis by introducing their cDNAs under a constitutive promoter. Overexpression of the cytochromecand hinge protein mRNAs was confirmed by Northern blotting, although marked accumulation of the cytochromecprotein was not observed. In transfectants of the hinge protein gene as well as cytochromecgene, apoptosis was accelerated as judged by FITC-conjugated Annexin V binding to the cell surface and DNA fragmentation. In addition, enhancement of the release of cytochromecinto cytosol was demonstrated in these transfectants by a subcellular fractionation experiment, followed by Western blotting. These findings suggest that the release of the cytochromecprotein from mitochondria is regulated by the hinge protein involved in the respiratory chain in the apoptotic process.

Identification of cDNA clones expressed selectively during the critical period for visual cortex development by subtractive hybridization

We have used the method of subtractive hybridization to isolate cDNA clones of mRNAs expressed in abundance in the visual cortex of 30-day-old kittens but absent or in lower abundance in the adult cat visual cortex. Of 12,000 colonies screened, 200 clones which hybridized to the subtracted probe were isolated and characterized. Northern blots confirmed the specificity of the vast majority of the isolated clones. 120 of the 200 clones were sequenced and the EMBL and GenBank (release 76) databases were searched for known identities using FASTA and BLAST programs. Twenty-seven of these sequenced clones were identifiable. The identities showed that these sequences code for proteins involved in a variety of cellular processes. These include cell-cell interaction (TAPA-1, contactin, tachykinin receptor, phospholipase A2), cellular remodeling (C1q beta isoform, heat shock protein), neurofilament assembly (alpha tubulin and alpha internexin), neurotransmitter release (VAMP-2, amphiphysin, car☐ypeptidase E, scg 10 and proton channel), energy metabolism (mitochondrial hinge protein, ADP/ATP transporter, cytochrome oxidase subunits), RNA processing (helix destabilizing protein, ribonucleoprotein) and protein synthesis (eIF-4A initiation factor, ribosomal protein S27). The results show that gene expression in the kitten visual cortex differs rather little from that of the adult visual cortex since over 98% of the sequences appear common. The relatively rare kitten-specific sequences are likely to form the basis for the critical period plasticity in this system.