Prohibitin Protein Research Articles

Emodin Improves Glucose and Lipid Metabolism Disorders in Obese Mice via Activating Brown Adipose Tissue and Inducing Browning of White Adipose Tissue.

BackgroundAdipose tissue (e.g. white, brown and brite) plays a critical role in modulating energy metabolism. Activating brown adipose tissue (BAT) and inducing browning in white adipose tissue (WAT) has been proposed to be a potential molecular target for obesity treatment. Emodin is a natural anthraquinone derivative that exhibits variety of pharmacologic effects including lowering lipids and regulating glucose utilization. However, the underlying mechanism of action is still unclear. In the present study, we investigated whether emodin could alleviate obesity via promoting browning process in adipose tissue.MethodsC57BL/6J mice were fed with high fat diet to induce obesity. Emodin at the doses of 40 and 80 mg/kg were orally given to obesity mice for consecutive 6 weeks. Parameters including fasting blood glucose, oral glucose tolerance, blood lipids, and the ratios of subcutaneous white adipose tissue (scWAT) or BAT mass to body weight, and morphology of adipose tissue were observed. Besides, the protein expression of uncoupling protein 1 (UCP1) and prohibitin in BAT and scWAT was determined by immunohistochemistry method. Relative mRNA expression of Cd137, transmembrane protein 26 (Tmem26) and Tbx1 in scWAT was analyzed using qRT-PCR. And the protein expression of UCP1, CD36, fatty acid transporter 4 (FATP4), peroxisome proliferator-activated receptor alpha (PPARα) and prohibitin of scWAT and BAT were analyzed using western blotting. In addition, ultra-high-performance liquid chromatography with electrospray ionization tandem mass spectrometry was utilized to detect the small lipid metabolites of scWAT and BAT.ResultsEmodin decreased the body weight and food intake in HFD-induced obesity mice, and it also improved the glucose tolerance and reduced the blood lipids. Emodin treatment induced beiging of WAT, and more multilocular lipid droplets were found in scWAT. Also, emodin significantly increased markers of beige adipocytes, e.g. Cd137, Tmem26 and Tbx1 mRNA in scWAT, and UCP1, CD36, FATP4, PPARα and prohibitin protein expression in scWAT and BAT. Furthermore, emodin perturbed the lipidomic profiles in scWAT and BAT of obese mice. Emodin increased total ceramides (Cers), lysophosphatidylcholines (LPCs), lyso-phosphatidylcholines oxygen (LPCs-O), and phosphatidylethanolamines oxygen (PEs-O) species concentration in scWAT. Specifically, emodin significantly up-regulated levels of Cer (34:1), LPC (18:2), LPC-(O-20:2), PC (O-40:7), PE (O-36:3), PE (O-38:6), PE (O-40:6), and sphingolipid (41:0) [SM (41:0)], and down-regulated PC (O-38:0), PE (O-40:4), PE (O-40:5) in scWAT of obesity mice. In terms of lipid matabolites of BAT, the emodin remarkably increased the total PCs levels, which was driven by significant increase of PC (30:0), PC (32:1), PC (32:2), PC (33:4) and PC (38:0) species. In addition, it also increased species of LPCs, e.g. LPC (20:0), LPC (20:1), LPC (22:0), LPC (22:1), LPC (24:0), and LPC (24:1). Especially, emodin treatment could reverse the ratio of PC/PE in HFD-induced obese mice.ConclusionsThese results indicated that emodin could ameliorate adiposity and improve metabolic disorders in obese mice. Also, emodin could promote browning in scWAT and activate the BAT activities. In addition, emodin treatment-induced changes to the scWAT and BAT lipidome were highly specific to certain molecular lipid species, indicating that changes in tissue lipid content reflects selective remodeling in scWAT and BAT of both glycerophospholipids and sphingolipids in response to emodin treatment.

Characterization and functional analysis of the proteins Prohibitin 1 and 2 in Trypanosoma cruzi.

BackgroundChagas disease is the third most important neglected tropical disease. There is no vaccine available, and only two drugs are generally prescribed for the treatment, both of which with a wide range of side effects. Our study of T. cruzi PHBs revealed a pleiotropic function in different stages of the parasite, participating actively in the transformation of the non-infective replicative epimastigote form into metacyclic trypomastigotes and also in the multiplication of intracellular amastigotes.Methodology/principal findingsTo obtain and confirm our results, we applied several tools and techniques such as electron microscopy, immuno-electron microscopy, bioinformatics analysis and molecular biology. We transfected T. cruzi clones with the PHB genes, in order to overexpress the proteins and performed a CRISPR/Cas9 disruption to obtain partially silenced PHB1 parasites or completely silenced PHB2 parasites. The function of these proteins was also studied in the biology of the parasite, specifically in the transformation rate from non-infective forms to the metacyclic infective forms, and in their capacity of intracellular multiplication.Conclusion/significanceThis research expands our understanding of the functions of PHBs in the life cycle of the parasite. It also highlights the protective role of prohibitins against ROS and reveals that the absence of PHB2 has a lethal effect on the parasite, a fact that could support the consideration of this protein as a possible target for therapeutic action.

Prohibitin regulates mTOR pathway via interaction with FKBP8.

The ability of tumor cells to sustain continuous proliferation is one of the major characteristics of cancer. The activation of oncogenes and the mutation or inactivation of tumor suppressor genes ensure the rapid proliferation of tumor cells. The PI3K-Akt-mTOR axis is one of the most frequently modified signaling pathways whose activation sustains cancer growth. Unsurprisingly, it is also one of the most commonly attempted targets for cancer therapy. FK506 binding protein 8 (FKBP8) is an intrinsic inhibitor of mTOR kinase that also exerts an anti-apoptotic function. We aimed to explain these contradictory aspects of FKBP8 in cancer by identifying a "switch" type regulator. We identified through immunoprecipitation-mass spectrometry-based proteomic analysis that the mitochondrial protein prohibitin 1 (PHB1) specifically interacts with FKBP8. Furthermore, the downregulation of PHB1 inhibited the proliferation of ovarian cancer cells and the mTOR signaling pathway, whereas the FKBP8 level in the mitochondria was substantially reduced. Moreover, concomitant with these changes, the interaction between FKBP8 and mTOR substantially increased in the absence of PHB1. Collectively, our finding highlights PHB1 as a potential regulator of FKBP8 because of its subcellular localization and mTOR regulating role.

Prohibitin, STAT3 and SH2D4A physically and functionally interact in tumor cell mitochondria

Chromosome 8p is frequently deleted in various cancer entities and has been shown to correlate with poor patient survival. SH2D4A is located on chromosome 8p and prevents the nuclear translocation of the pro-tumorigenic transcription factor STAT3. Here, we investigated the interaction of SH2D4A and STAT3 to shed light on the non-canonical functions of STAT3 in cooperation with the tumor suppressor SH2D4A. Using an immunoprecipitation-mass spectrometry (IP-MS) approach, we identified the mitochondrial scaffold proteins prohibitin 1 (PHB1) and prohibitin 2 (PHB2) among other proteins to potentially bind to SH2D4A. Co-immunoprecipitation and proximity ligation assays confirmed direct interactions of STAT3, PHB1, and SH2D4A in situ and in vitro. In addition, cell fractionation and immunofluorescence staining revealed co-localization of these proteins with mitochondria. These interactions were selectively interrupted by the small molecule and PHB ligand FL3. Furthermore, FL3 led to a reduction of STAT3 protein levels, STAT3 transcriptional activity, and HIF1α protein stabilization upon dimethyloxalylglycine (DMOG) treatment. Besides, mitochondrial fusion and fission markers, L-OPA1, Mfn1, and FIS1, were dysregulated upon FL3 treatment. This dysregulated morphology was accompanied by significant reduction of mitochondrial respiration, thus, FL3 significantly diminished mitochondrial respirational capacity. In contrast, SH2D4A knockout increased mitochondrial respiration, whereas FL3 reversed the effect of SH2D4A knockout. The here described results indicate that the interaction of SH2D4A and PHB1 is involved in the mitochondrial function and integrity. The demonstrated interaction with STAT3, accompanied by its reduction of transcriptional activity, further suggests that SH2D4A is linking STAT3 to its mitochondrial functions, and inhibition of PHB-interaction may have therapeutic effects in tumor cells with STAT3 activation.

Mitochondrial dysfunction promotes aquaporin expression that controls hydrogen peroxide permeability and ferroptosis

Most anti-cancer agents and radiotherapy exert their therapeutic effects via the production of free radicals. Ferroptosis is a recently described cell death process that is accompanied by iron-dependent lipid peroxidation. Hydrogen peroxide (H2O2) has been reported to induce cell death. However, it remains controversial whether H2O2-induced cell death is ferroptosis. In the present study, we aimed to elucidate the involvement of mitochondria in H2O2-induced ferroptosis and examined the molecules that regulate ferroptosis. We found that one mechanism underlying H2O2-induced cell death is ferroptosis, which occurs soon after H2O2 treatment (within 3 h after H2O2 treatment). We also investigated the involvement of mitochondria in H2O2-induced ferroptosis using mitochondrial DNA-depleted ρ0 cells because ρ0 cells produce more lipid peroxidation, hydroxyl radicals (•OH), and are more sensitive to H2O2 treatment. We found that ρ0 cells contain high Fe2+ levels that lead to •OH production by H2O2. Further, we observed that aquaporin (AQP) 3, 5, and 8 bind nicotinamide-adenine dinucleotide phosphate oxidase 2 and regulate the permeability of extracellular H2O2, thereby contributing to ferroptosis. Additionally, the role of mitochondria in ferroptosis was investigated using mitochondrial transfer in ρ0 cells. When mitochondria were transferred into ρ0 cells, the cells exhibited no sensitivity to H2O2-induced cytotoxicity because of decreased Fe2+ levels. Moreover, mitochondrial transfer upregulated the mitochondrial quality control protein prohibitin 2 (PHB2), which contributes to reduced AQP expression. Our findings also revealed the involvement of AQP and PHB2 in ferroptosis. Our results indicate that H2O2 treatment enhances AQP expression, Fe2+ level, and lipid peroxidation, and decrease mitochondrial function by downregulating PHB2, and thus, is a promising modality for effective cancer treatment.

Expression and co-location of prohibitin genes in salt-tolerance QTLs in rice (Oryza sativa)

Prohibitin proteins are involved in diverse biological functions including cell cycle regulation, mitochondrial activity, apoptosis, hormonal signalling and so on. Prohibitin family genes are conserved across the life taxa in both eukaryotes and prokaryotes. An experiment was conducted at ICAR-National Institute for Plant Biotechnology, New Delhi (2018-19) in which we identified 19 prohibitin genes present on 10 different chromosomes of the ‘Nipponbare’ reference rice (Oryza sativa L.) genome and analysed their possible role in salt tolerance, as they are known to regulate ion channels in plant and animal cells. A protein sequence based neighbour joining phylogentic tree clustered these 19 genes into three groups. Digital gene expression analysis revealed the highest level of expression of the prohibitin genes in anthers and radicle tips. Nine of these prohibitin genes were co-located in the salt-tolerance QTL regions of the rice genome, three of which (LOC_Os01g40560, LOC_Os03g60000 and LOC_Os08g15600) were significantly upregulated in response to salt stress in the tolerant genotype. These prohibitin genes are strong candidates for improving salt-tolerance in rice through marker-assited rice breeding after validation.

Prohibitin‐1 plays a multifaceted role in mediating luteinizing hormone‐induced steroidogenesis in Leydig cells.

Leydig cells (LCs) present in the interstitium between seminiferous tubules in the testis are responsible for the biosynthesis of testosterone from cholesterol in response to luteinizing hormone (LH) from the pituitary. The principal pathways of testosterone biosynthesis, the identities of the main steroidogenic enzymes, and genetic disorders of most of these enzymes have been elucidated. However, many important questions related to LH signaling and cholesterol transport remain unknown. For example, the mechanisms of StAR‐mediated cholesterol transport from the outer mitochondrial membrane to cytochrome P450 side chain cleavage (P450scc or Cyp11a1) enzyme in the inner mitochondrial membrane, which is a critical step in steroidogenesis, remain elusive. It is conceivable that we are missing some important players involved in this process.Prohibitin‐1 (PHB1) is a pleiotropic protein with cell compartment‐specific functions, including the phosphorylation‐dependent membrane signaling and mitochondrial chaperone, which appears to require heterodimerization with its homologous protein prohibitin‐2 (PHB2) in the inner mitochondrial membrane. Previously, we have reported transgenic mouse models expressing PHB1 (PHB‐Tg) or Y114F‐PHB1 (mPHB‐Tg) from the fatty acid binding protein‐4 (Fabp4) gene promoter. During their phenotypic characterization, unexpectedly, we found that male mPHB‐Tg mice, but not PHB‐Tg mice, have high testosterone levels. Subsequent analysis revealed that it was due to leaky overexpression of m/PHB1 in LCs. This prompted us to investigate whether LH regulates PHB1 in LCs. A dose and time‐dependent effect of human chorionic gonadotropin, hCG (pituitary analog of LH) was found on PHB1 expression levels in LCs. To define the cell compartment‐specific role of PHB1 in LCs, we investigated the functional status of LH signaling in testes/LCs and ultrastructure of LCs from transgenic mice. A difference in phospho‐ERK levels was found between PHB‐Tg and mPHB‐Tg, which was higher in mPHB1 expressing cells. A similar effect was found in PHB1 manipulated MA‐10 cells (a model LC line). A parallel change in ultrastructural features of LCs (mitochondrial structure and lipid droplets) was found in PHB and mPHB overexpressing cells. Moreover, co‐immunoprecipitation experiments showed that PHB1 and PHB2 interact with P450scc and/or StAR. Analysis of PHBs protein sequences revealed the presence of multiple CRAC and/or CARC motifs (cholesterol recognition motifs) in both proteins. In aggregate, this finding suggests that PHB1 is a LH regulated protein in LCs and plays a multifaceted role in LH‐induced steroidogenesis, including ERK activation and potentially in the functional coupling of StAR and P450scc across mitochondrial membrane for steroidogenesis. The scope of our findings is broad because the basics of cholesterol transport across mitochondrial membranes are similar in different steroidogenic tissues.

Peptidylarginine Deiminase Isozyme-Specific PAD2, PAD3 and PAD4 Inhibitors Differentially Modulate Extracellular Vesicle Signatures and Cell Invasion in Two Glioblastoma Multiforme Cell Lines.

Glioblastoma multiforme (GBM) is an aggressive adult brain tumour with poor prognosis. Roles for peptidylarginine deiminases (PADs) in GBM have recently been highlighted. Here, two GBM cell lines were treated with PAD2, PAD3 and PAD4 isozyme-specific inhibitors. Effects were assessed on extracellular vesicle (EV) signatures, including EV-microRNA cargo (miR21, miR126 and miR210), and on changes in cellular protein expression relevant for mitochondrial housekeeping (prohibitin (PHB)) and cancer progression (stromal interaction molecule 1 (STIM-1) and moesin), as well as assessing cell invasion. Overall, GBM cell-line specific differences for the three PAD isozyme-specific inhibitors were observed on modulation of EV-signatures, PHB, STIM-1 and moesin protein levels, as well as on cell invasion. The PAD3 inhibitor was most effective in modulating EVs to anti-oncogenic signatures (reduced miR21 and miR210, and elevated miR126), to reduce cell invasion and to modulate protein expression of pro-GBM proteins in LN229 cells, while the PAD2 and PAD4 inhibitors were more effective in LN18 cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for deiminated proteins relating to cancer, metabolism and inflammation differed between the two GBM cell lines. Our findings highlight roles for the different PAD isozymes in the heterogeneity of GBM tumours and the potential for tailored PAD-isozyme specific treatment.

Characterisation, expression and possible functions of prohibitin during spermatogenesis in the silver pomfret Pampus argenteus.

Mitochondria play an important role in spermatogenesis, and some mitochondrial proteins are specifically related to this process. In this study we investigated the cytological characteristics of spermatogenic cells, including mitochondrial dynamics, during spermatogenesis in Pampus argenteus. In addition, we characterised the mitochondria-related protein prohibitin (PHB), which has been reported to play roles in mitochondrial dynamics and animal fertility. The full-length cDNA of the P. argenteus phb gene (Pa-phb) is 1687bp, including a 102-bp 5'-untranslated region (UTR), a 772-bp 3'-UTR and an 813-bp open reading frame encoding 271 amino acids. The predicted P. argenteus PHB protein (Pa-PHB) contains three functional domains (a transmembrane domain, an SPFH domain (the conserved region of stomatins, prohibitins, flotillins and HflK/C) and a coiled-coil domain) and exhibits high similarity with its homologue in other animals. The Pa-phb gene was widely expressed in all tissues examined, especially the liver and heart. We primarily focused on Pa-phb expression during spermatogenesis after observing the cytological features of male germ cells, and found that Pa-phb transcripts were detected throughout the course of development of male germ cells. Notably, we observed colocalised signals of Pa-PHB and mitochondria, which were distributed in the cytoplasm around the nucleus in spermatogonia, spermatocytes and early spermatids, tended to move to one side of the cell in middle spermatids and, finally, were colocalised in the sperm midpiece. These observations indicate that Pa-PHB is primarily localised in mitochondria during spermatogenesis, indicating that it has a role in mitochondria. Based on the results of this and previous studies regarding the essential roles of PHB in mitochondria and spermatogenesis in animals, we propose a functional model for PHB during spermatogenesis, including possible roles in the proliferation of spermatogonia and in the regulation of mitochondrial morphology and function in spermatogenic cells.

Characterization of PHB in the gonadal development of the swimming crab Portunus trituberculatus.

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. In this study, we cloned the PHB gene from the testis of the swimming crab Portunus trituberculatus (PtPHB) and analyzed the deduced amino acid sequence. The expression level of phb mRNA in larvae was analyzed using qRT-PCR. The expression level of phb mRNA and PHB protein in different tissues were analyzed using qRT-PCR and Western blot respectively. Enzyme-linked immunosorbent assay analyses of the PHB protein were conducted with the testis and ovaries from P. trituberculatus specimens at different developmental stages. PHB was localized with mitochondria and ubiquitin in the testis and ovaries. The PtPHB gene was found to contain an open reading frame of 825 bp, encoding a predicted peptide with 275 amino acids, sharing between 65.9% and 96.7% similarity with that of other species. The qRT-PCR and Western blot results showed that the phb gene and PHB protein both expressed less in the testis and ovary than in other tissues, and the phb gene presented the lowest expression in the Z1 stage. Furthermore, the phb gene and PHB protein expression were different in the testis and ovaries at different developmental stages. PHB was mainly found to be co-localized with mitochondria and ubiquitin in cytoplasm and acrosome complex during spermatogenesis and in follicular cells during oogenesis. Interestingly, PHB-mitochondria signals and ubiquitin signal were also found in oocytes. These results indicated that PHB might play important roles during spermatogenesis and oogenesis by regulating mitochondrial activities.

NAD+ repletion inhibits the endothelial-to-mesenchymal transition induced by TGF-β in endothelial cells through improving mitochondrial unfolded protein response.

Endothelial-to-mesenchymal transition (EndMT) plays an important role in the progression of cardiac fibrosis but its mechanism and treatment need to be further understood. Herein, we have found that mitochondrial unfolded protein response (mtUPR) played a critical role in transforming growth factor beta 1 (TGF-β1)-induced EndMT in endothelial cells (ECs). MtUPR was repressed in endothelial cells after exposure to TGF-β1. NAD + precursor nicotinamide riboside (NR) could attenuate TGF-β1-induced EndMT and improve the levels of mtUPR. Significantly, prohibitin proteins (PHB and PHB2) was also regulated by nicotinamide riboside. Moreover, we found that inhibition of prohibitin proteins could prevent the protective effect of nicotinamide riboside on mtUPR and TGF-β1-induced EndMT. Overexpression of prohibitin proteins could alleviate mitochondrial function and TGF-β1-induced EndMT through improving mtUPR. In vivo, The EndMT of ECs induced by Transverse aortic constriction (TAC) in mouse was inhibited by NR. In conclusion, our results indicate that nicotinamide riboside improved the expression of prohibitin proteins to ameliorate EndMT via promotion of mtUPR. Nicotinamide riboside is a potential therapeutic target for cardiac fibrosis.

Effects of prohibitin on ageing process and reproduction ofDaphnia pulex

Daphnia pulex is considered as a great natural food for aquaculture, and Prohibitin (PHB) is a functional protein that plays a key role in eukaryotic cell activities such as proliferation and ageing. To explore the function of PHB in the ageing process of Daphnia pulex (D. pulex), we cloned and analysed the full-length cDNA of phb1 and phb2. With the growth of D. pulex, expression of two genes have a N curve trend which raised up first and dropped then raised again. Western blot analysis revealed that expression of the PHB proteins increased from 1- to 10-day age, but did not significantly change from 10- to 35-day age. RNAi knockdown of phb1, reduced expression of phb2 and PHB protein, and significantly induced the expression of sex-determining related gene Doublesex (Dsx), and ageing-related pathway gene Protein kinase B (Akt). Longevity of the individuals in RNAi group was shortened to 45 days, which was significantly shorter than the other groups. Despite the growth was not significantly different, reproduction amount was significantly impacted by RNAi. These results suggest that PHB significantly affect ageing of D. pulex and may be involved in the reproductive transformation, leading to a decline of reproductive capacity. This study develops our understanding of the ageing mechanism in D. pulex as a potential model organism, provides further insight into the effect of PHB in the processes of ageing and expands our knowledge of molecular mechanisms on ageing and reproduction.

All-trans retinoic acid regulated prohibitin by retinoic acid receptor α in hypoxia-induced renal tubular epithelial cell injury

All-trans retinoic acid (ATRA) is a critical component in cell processes such as cell growth, differentiation and apoptosis, and it is also crucial in the regulation of extracellular matrix (ECM) deposition. Prohibitin (PHB) can regulate cell proliferation, apoptosis and differentiation. The current study investigated whether ATRA regulated PHB is induced by hypoxia/reoxygenation injury in renal tubular epithelial cells (RTEC), using gene interference treatments (knockdown or overexpression of RARα). Our results indicate that ATRA can augment the expression of RARα and PHB proteins and reduce the expression of TGF-β1, FN and Col-IV proteins. PHB expression was reduced in an ATRA treated RARα- group, and TGF-β1, FN and Col-IV were up-regulated compared to the ATRA treated RARα+ group. We postulate that ATRA can induce the PHB expression by RARα in hypoxia/reperfusion related RTEC injury.

Immunodiagnosis of human and canine visceral leishmaniasis using recombinant Leishmania infantum Prohibitin protein and a synthetic peptide containing its conformational B-cell epitope

In the present study, Leishmania infantum's Prohibitin was cloned and, alongside a synthetic peptide, evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis (CVL and TL, respectively) in dogs and humans. For TL diagnosis, this study analyzed serum samples from cutaneous (n = 20) or mucosal (n = 39) leishmaniasis patients, and from Chagas disease (CD) patients (n = 8) and non-infected patients (n = 45). For CVL diagnosis, serum samples from asymptomatic (n = 14), symptomatic (n = 71), non-infected (n = 116), and Leish-Tec®-vaccinated (n = 79) dogs were examined, as well as T. cruzi (n = 11) and Ehrlichia canis (n = 10) infected animals. An indirect ELISA method using rProhibitin showed diagnostic sensitivity and specificity values of 91.76% and 89.91%, respectively. L. infantum SLA showed 86.11% and 48.24% of specificity and sensitivity, respectively, for CVL serodiagnosis, and 98.31% and 84.91% sensitivity and specificity, respectively for TL diagnosis. L. braziliensis SLA showed 75.47% and 83.05% of specificity and sensitivity, respectively, for TL diagnosis. The synthetic peptide showed a better result in TL than in CVL diagnosis. In conclusion, preliminary results suggest that the detection of antibodies against the rProhibitin protein and the synthetic peptide improves the serodiagnosis of TL and CVL.

Antibodies against H1N1 influenza virus hemagglutinin cross-react with prohibitin

Influenza virus infection is associated with type 1 diabetes (T1DM), but its pathogenesis remains unclear. Here, our study found that one of the monoclonal antibodies against H1N1 influenza virus hemagglutinin(HA) cross-reacted with human pancreatic tissue and further demonstrated that it binded to rat islet β-cells. We immunoprecipitated islet protein with this cross-reactive antibody and identified the bound antigen as prohibitin by mass spectrometry. We then expressed the prohibitin protein in bacteria and confirmed the antibody binding to prohibitin by Western blot. We also verified the cross-reactivity of the antibody by prohibitin-siRNA transfection in islet beta cells. We conclude that prohibitin is an autoantigen that cross-reacts with influenza virus HA. The correlation between the autoantigen prohibitin and type 1 diabetes remains to be investigated.

Prohibitin Shuttles Between Mitochondria and the Nucleus to Control Genome Stability During the Cell Cycle.

The prohibitin (PHB) protein family is highly conserved from prokaryotes to eukaryotes ([Van Aken et al., 2010][1]). Initially identified as a tumor suppressor in rat liver, PHBs in animals are localized in various cell compartments, including mitochondria, nuclei, and plasma membranes, and function

The mechanism of overexpression of retinoic acid receptor alpha in attenuating renal interstitial fibrosis in rats

Objective To study the mechanism of overexpression of retinoic acid receptor alpha (RARα) in attenuating renal interstitial fibrosis (RIF) in rats. Methods Forty 6-week-old male SD rats were randomly divided into 4 groups: sham operation group, model group, negative control group and transfection group, with 10 rats in each group.Rats in model group were separated and double ligated with left ureter; rats in sham operation group were not ligated with ureter; rats in transfection group and negative control group were transfected with adeno-associated virus and negative control virus carrying RARα gene on the basis of model group, respectively.All rats were sacrificed 2 weeks later.Left kidney tissues were taken for pathological examination and RIF index was calculated.The expression of collagen Ⅳ (Col-Ⅳ) and fibronectin (FN) in renal tissue was detected by using immunohistochemistry.The expressions of RARα, prohibitin (PHB) and transforming growth factor-beta 1 (TGF-β1) in renal tissue were detected by using real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) and Western blot. Results (1)Compared with sham operation group, the RIF index was significantly increased in model group (22.81±2.43 vs.2.34±0.55, q=24.94, P 0.05), however it significantly decreased in transfection group (14.06±2.99 vs.22.81±2.43, q=10.66, P 0.05); compared with model group, the mRNA and protein expression of RARα, PHB mRNA and protein expression significantly increased, but the TGF-β1 mRNA and protein, Col-Ⅳ and FN protein expression significantly decreased in transfected group (mRNA: 0.86±0.07 vs.0.43±0.17, 0.89±0.11 vs.0.34±0.08, 1.65±0.28 vs.2.97±0.54, all P<0.05; protein: 0.40±0.07 vs.0.25±0.10, 0.45±0.10 vs.0.24±0.07, 0.43±0.08 vs.0.59±0.09, 11.57±0.33 vs.16.01±0.87, 11.67±0.53 vs.14.64±0.32, all P<0.05). (3)Correlation analysis revealed that RARα protein expression was negatively correlated with RIF index, Col-Ⅳ, FN, TGF-β1 (r=-0.78, -0.78, -0.76, -0.76, all P<0.05); PHB protein expression was negatively correlated with RIF index, Col-Ⅳ, FN, TGF-β1 (r=-0.87, -0.87, -0.88, -0.75, all P<0.05); RARα protein was positively correlated with PHB (r=0.85, P<0.05). Conclusion Overexpression of RARα could attenuate RIF by enhancing PHB expression in rats subjected to unilateral ureteral obstruction. Key words: Retinoic acid receptor alpha; Prohibitin; Renal interstitial fibrosis; Transfection

Estrogen Enhances Endometrial Cancer Cells Proliferation by Upregulation of Prohibitin.

Estrogen plays an essential role in type I endometrial cancer cell proliferation. Despite great progresses in the etiology has been obtained in the past, however, the molecular mechanisms remain to be fully clarified. Prohibitin has been demonstrated involvement in multiple cancers' development. If it also contributes to estrogen-driven endometrial cancer proliferation is not clear. IHC assay result display that prohibitin overexpressed in endometrial cancer tissue and associated with the poor prognosis; Western blot assay detect that upregulated prohibitin expression with dose- and time-dependent manners. The cellular growth was monitored with SRB assay which demonstrate that knockdown prohibitin attenuated estrogen-induced proliferation. Ubiquitination assay finds estrogen increased prohibitin level through stabilizing prohibitin protein via inhibition of ubiquitination, while estrogen-induced protein expression was mediated by estrogen receptor. Our findings provide a new insight on the mechanism of estrogen-induced proliferation, implying the possibility of using prohibitin as a potential therapeutic target for the treatment of endometrial cancer.

Cannabidiol Affects Extracellular Vesicle Release, miR21 and miR126, and Reduces Prohibitin Protein in Glioblastoma Multiforme Cells

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary malignant brain tumor in adults, with poor prognosis. Extracellular vesicles (EVs) are key-mediators for cellular communication through transfer of proteins and genetic material. Cancers, such as GBM, use EV release for drug-efflux, pro-oncogenic signaling, invasion and immunosuppression; thus the modulation of EV release and cargo is of considerable clinical relevance. As EV-inhibitors have been shown to increase sensitivity of cancer cells to chemotherapy, and we recently showed that cannabidiol (CBD) is such an EV-modulator, we investigated whether CBD affects EV profile in GBM cells in the presence and absence of temozolomide (TMZ). Compared to controls, CBD-treated cells released EVs containing lower levels of pro-oncogenic miR21 and increased levels of anti-oncogenic miR126; these effects were greater than with TMZ alone. In addition, prohibitin (PHB), a multifunctional protein with mitochondrial protective properties and chemoresistant functions, was reduced in GBM cells following 1 h CBD treatment. This data suggests that CBD may, via modulation of EVs and PHB, act as an adjunct to enhance treatment efficacy in GBM, supporting evidence for efficacy of cannabinoids in GBM.

Peptidylarginine Deiminases Post-Translationally Deiminate Prohibitin and Modulate Extracellular Vesicle Release and MicroRNAs in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most aggressive form of adult primary malignant brain tumour with poor prognosis. Extracellular vesicles (EVs) are a key-mediator through which GBM cells promote a pro-oncogenic microenvironment. Peptidylarginine deiminases (PADs), which catalyze the post-translational protein deimination of target proteins, are implicated in cancer, including via EV modulation. Pan-PAD inhibitor Cl-amidine affected EV release from GBM cells, and EV related microRNA cargo, with reduced pro-oncogenic microRNA21 and increased anti-oncogenic microRNA126, also in combinatory treatment with the chemotherapeutic agent temozolomide (TMZ). The GBM cell lines under study, LN18 and LN229, differed in PAD2, PAD3 and PAD4 isozyme expression. Various cytoskeletal, nuclear and mitochondrial proteins were identified to be deiminated in GBM, including prohibitin (PHB), a key protein in mitochondrial integrity and also involved in chemo-resistance. Post-translational deimination of PHB, and PHB protein levels, were reduced after 1 h treatment with pan-PAD inhibitor Cl-amidine in GBM cells. Histone H3 deimination was also reduced following Cl-amidine treatment. Multifaceted roles for PADs on EV-mediated pathways, as well as deimination of mitochondrial, nuclear and invadopodia related proteins, highlight PADs as novel targets for modulating GBM tumour communication.