Prohibitin Research Articles

The Role of Prohibitin-2 in Diseases.

Prohibitin-2 (PHB2) is a conserved protein in mitochondria that regulates various biological processes, including cell cycle, proliferation, apoptosis, transcription, signal transduction, and mitochondrial ridge morphogenesis. Recently, there has been growing interest in the biological function of PHB2. This article primarily discusses the recent advances in the role of PHB2 in diseases.

Prohibitin-1 is an ACTH-Regulated Protein in Human and Mouse Adrenocortical Cells and Plays a Role in Corticosteroid Production

Cell-intrinsic early events involved in different trophic hormone-induced steroidogenesis in their respective steroidogenic cell type are very similar. For example, the activation of the cAMP-PKA signaling pathway in response to trophic hormone stimulation and, subsequently, cholesterol transport to the mitochondria to initiate steroidogenesis is common to them. Recently, we have found that an evolutionarily conserved protein, prohibitin-1 (PHB1), is regulated by Luteinizing Hormone (LH) in murine Leydig cells and plays a role in interconnected cell signaling and mitochondrial steps pertaining to testosterone production. Among the primary steroidogenic tissues, PHB1 expression levels are highest in the adrenal cortex (The Human Protein Atlas); however, its regulation and role in adrenocortical cells are virtually unknown. We investigated the regulation and the role of PHB1 in adrenocortical cells in vitro using human HAC15 and mouse Y-1 cell culture models. It was found that Adrenocorticotrophic Hormone (ACTH) stimulation upregulates PHB1 levels in adrenocortical cells in a time-dependent manner. A similar effect on PHB1 levels was also observed in response to dibutyryl-cAMP stimulation, a cell-permeable analogue of cAMP (the second messenger for ACTH action). Moreover, manipulating PHB1 levels in adrenocortical cells affected mitochondria, lysosomes, and lipid droplet characteristics, modulated phospho-PKA and phospho-ERK1/2 levels, and altered corticosteroid production. This finding suggests that ACTH regulates PHB1 in adrenocortical cells and plays a role in corticosteroid production, which was previously unknown.

Control of Paneth cell function by HuR regulates gut mucosal growth by altering stem cell activity.

Rapid self-renewal of the intestinal epithelium requires the activity of intestinal stem cells (ISCs) that are intermingled with Paneth cells (PCs) at the crypt base. PCs provide multiple secreted and surface-bound niche signals and play an important role in the regulation of ISC proliferation. Here, we show that control of PC function by RNA-binding protein HuR via mitochondria affects intestinal mucosal growth by altering ISC activity. Targeted deletion of HuR in mice disrupted PC gene expression profiles, reduced PC-derived niche factors, and impaired ISC function, leading to inhibited renewal of the intestinal epithelium. Human intestinal mucosa from patients with critical surgical disorders exhibited decreased levels of tissue HuR and PC/ISC niche dysfunction, along with disrupted mucosal growth. HuR deletion led to mitochondrial impairment by decreasing the levels of several mitochondrial-associated proteins including prohibitin 1 (PHB1) in the intestinal epithelium, whereas HuR enhanced PHB1 expression by preventing microRNA-195 binding to the Phb1 mRNA. These results indicate that HuR is essential for maintaining the integrity of the PC/ISC niche and highlight a novel role for a defective PC/ISC niche in the pathogenesis of intestinal mucosa atrophy.

Genistein enhances NAD+ biosynthesis by upregulating nicotinamide phosphoribosyltransferase in adipocytes

A decrease in the NAD+ level in adipocytes causes adipose-tissue dysfunction, leading to systemic glucose, and lipid metabolism failure. Therefore, it is necessary to develop small molecules and nutraceuticals that can increase NAD+ levels in adipocytes. Genistein, a nutraceutical derived from soybeans, has various physiological activities and improves glucose and lipid metabolism. In this study, we aimed to unravel the effects of genistein on the NAD+ level in adipocytes and the underlying molecular mechanisms. Genistein enhanced NAD+ biosynthesis by increasing the expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. A pull-down assay using genistein-immobilized beads revealed prohibitin 1 (PHB1) as a target protein of genistein. The knockdown of Phb1 suppressed the genistein-induced increase in NAMPT expression and NAD+ level in adipocytes. Genistein-bound PHB1 contributed to the stabilization of the transcription factor CCAAT/enhancer-binding protein β through the activation of extracellular signal-regulated kinase, resulting in increased NAMPT expression at the transcriptional level. Genistein induced the dephosphorylation of peroxisome proliferator-activated receptor at serine 273 and increased the level of the insulin-sensitizing adipokine adiponectin in adipocytes, whereas the knockdown of Nampt and Phb1 abolished these genistein-mediated effects. Our results proved the potential efficacy of genistein in increasing the NAD+ level and restoring metabolic function in adipocytes. Furthermore, we identified PHB1, localized to the plasma membrane, as a novel candidate target protein for increased expression of NAMPT in adipocytes. Overall, these findings will assist in developing NAD+-boosting nutraceuticals to alleviate metabolic dysfunctions in adipose tissues.

Flt3 Activation Mitigates Mitochondrial Fragmentation and Heart Dysfunction through Rebalanced L-OPA1 Processing by Hindering the Interaction between Acetylated p53 and PHB2 in Cardiac Remodeling.

Recent studies have shown that FMS-like receptor tyrosine kinase 3 (Flt3) has a beneficial effect on cardiac maladaptive remodeling. However, the role and mechanism of Flt3 in mitochondrial dynamic imbalance under cardiac stress remains poorly understood. This study aims to investigate how Flt3 regulates p53-mediated optic atrophy 1 (OPA1) processing and mitochondrial fragmentation to improve cardiac remodeling. Mitochondrial fragmentation in cardiomyocytes was induced by isoprenaline (ISO) and H2O2 challenge, respectively, in vitro. Cardiac remodeling in mice was established by ligating the left anterior descending coronary artery or by chronic ISO challenge, respectively, in vivo. Our results demonstrated that the protein expression of acetylated-p53 (ac-p53) in mitochondria was significantly increased under cell stress conditions, facilitating the dissociation of PHB2-OPA1 complex by binding to prohibitin 2 (PHB2), a molecular chaperone that stabilizes OPA1 in mitochondria. This led to the degradation of the long isoform of OPA1 (L-OPA1) that facilitates mitochondrial fusion and resultant mitochondrial network fragmentation. This effect was abolished by a p53 K371R mutant that failed to bind to PHB2 and impeded the formation of the ac-p53-PHB2 complex. The activation of Flt3 significantly reduced ac-p53 expression in mitochondria via SIRT1, thereby hindering the formation of the ac-p53-PHB2 complex and potentiating the stability of the PHB2-OPA1 complex. This ultimately inhibits L-OPA1 processing and leads to the balancing of mitochondrial dynamics. These findings highlight a novel mechanism by which Flt3 activation mitigates mitochondrial fragmentation and dysfunction through the reduction of L-OPA1 processing by dampening the interaction between ac-p53 and PHB2 in cardiac maladaptive remodeling.

Prohibitin2/PHB2, Transcriptionally Regulated by GABPA, Inhibits Cell Growth via PRKN/Parkin-dependent Mitophagy in Endometriosis.

Endometriosis (EMS) is a common benign gynecological disease affecting women of reproductive age. It is characterized by abnormal growth of endometrial tissue outside the uterine cavity, resulting in chronic pelvic pain and infertility. Endometrial physiological and pathological processes are intimately connected to autophagy. Mitophagy is an essential selective mode that protects cells from metabolic stress and hypoxia. Mitochondrial autophagy mediated by prohibitin 2 (PHB2) is dependent on the PRKN/Parkin pathway and is involved in numerous human diseases. Uncertainty remains as to whether mitophagy regulation by PHB2 contributes to the occurrence and progression of EMS. This study aims to investigate the mechanism underlying the role of PHB2 in EMS. This study detected the protein and mRNA expression of PHB2 in ectopic and normal endometrial tissues of ovarian EMS, in addition to ectopic endometrial cell line 12Z and endometrial stromal cell line KC02-44D for gene overexpression or knockdown. Cell function experiments and mitochondrial function experiments were conducted to investigate the role of PHB2 in the endometrium. Bioinformatic analysis and experiments were also used to investigate the upstream transcription factors that influence PHB2 expression. PHB2 was downregulated in ectopic endometrium, and PHB2 overexpression inhibited cell proliferation, migration, and invasion and promoted apoptosis. The upregulation of mitophagy markers, including Parkin and LC3II/I, and the downregulation of autophagy degradation markers P62 and TOMM20 in EMS suggest that PHB2 may contribute to cell proliferation, migration, invasion, and apoptosis via PRKN/Parkin-mediated mitophagy. Analysis and validation of bioinformatics data revealed that the transcription factor GABPA binds directly to the PHB2 promoter region and controls the transcriptional expression of PHB2. This study investigated the role of PHB2 in the onset of EMS. It inhibits EMS growth via PRKN/Parkin-mediated mitophagy, and GABPA controls the transcriptional disorder of PHB2. This study's findings suggest a novel method for investigating the clinical potential of PHB2 in EMS.

PHB2 ameliorates Doxorubicin-induced cardiomyopathy through interaction with NDUFV2 and restoration of mitochondrial complex I function

BackgroundDoxorubicin (DOX) is among the most widely employed antitumor agents, although its clinical applications have been largely hindered by severe cardiotoxicity. Earlier studies described an essential role of mitochondrial injury in the pathogenesis of DOX cardiomyopathy. PHB2 (Prohibitin 2) is perceived as an essential regulator for mitochondrial dynamics and oxidative phosphorylation (OXPHOS) although its involvement in DOX cardiomyopathy remains elusive. MethodsTo decipher the possible role of PHB2 in DOX cardiomyopathy, tamoxifen-induced cardiac-specific PHB2 conditional knockout mice were generated and subjected to DOX challenge. Cardiac function and mitochondrial profiles were examined. Screening of downstream mediators of PHB2 was performed using proteomic profiling and bioinformatic analysis, and was further verified using co-immunoprecipitation and pulldown assays. ResultsOur data revealed significantly downregulated PHB2 expression in DOX-challenged mouse hearts. PHB2CKO mice were more susceptible to DOX cardiotoxicity compared with PHB2flox/flox mice, as evidenced by more pronounced cardiac atrophy, interstitial fibrosis and decrease in left ventricular ejection fraction and fractional shortening. Mechanistically, PHB2 deficiency resulted in the impairment of mitochondrial bioenergetics and oxidative phosphorylation in DOX cardiotoxicity. Proteomic profiling and interactome analyses revealed that PHB2 interacted with NDUFV2 (NADH-ubiquinone oxidoreductase core subunit V2), a key subunit of mitochondrial respiratory Complex I to mediate regulatory property of PHB2 on mitochondrial metabolism. PHB2 governed the expression of NDUFV2 by promoting its stabilization, while PHB2 deficiency significantly downregulated NDUFV2 in DOX-challenged hearts. Cardiac overexpression of PHB2 alleviated mitochondrial defects in DOX cardiomyopathy both in vivo and in vitro. ConclusionsOur study defined a novel role for PHB2 in mitochondrial dynamics and energetic metabolism through interacting with NDUFV2 in DOX-challenged hearts. Forced overexpression of PHB2 may be considered a promising therapeutic approach for patients with DOX cardiomyopathy.

Investigation of HER2 I655V and PHB 3′UTR C > T polymorphisms in azoospermic infertile males

BackgroundMale infertility is a complex, multifactorial pathological condition with a highly heterogeneous phenotypic variation, from complete absence of spermatozoa in the testicles (azoospermia) to marked changes in sperm quality. The Erb-B2 receptor tyrosine kinase 2 gene (ERBB2, also often called HER2) was hypothesized to be involved in spermatogenesis and Leydig cell steroidogenesis in males. Prohibitin (PHB) has been shown to regulate sperm motility in infertile men by changing mitochondrial membrane potential and increasing reactive oxygen species levels. In this study, we aimed to investigate the HER2 I655V and PHB 3′UTR C > T polymorphisms in azoospermic infertile males.MethodsOne hundred and thirty-three infertile males with idiopathic azoospermia and 100 healthy male controls were included in this study. Patient and control DNAs, which were extracted from peripheral blood, were analyzed using polymerase chain reaction and restriction fragment length polymorphism technics. For statistical evaluation, Chi-square and variance analyses were carried out using IBM SPSS (version 20.0) and Openepi (version 3.01) software programs.ResultsWe did not observe any differences between azoospermic infertile males and healthy male controls in terms of allele and genotype distributions of both the HER2 I655V and PHB 3′UTR C > T polymorphisms (p > 0.05). Moreover, composite genotype analyses did not show any differences between two groups (p > 0.05).ConclusionsNo association was found between HER2 I655V and PHB 3′UTR C > T polymorphisms and azoospermic male infertility. The study can be expanded further by increasing the number of samples and studying in various populations.

The Effect of Astaxanthin on Mitochondrial Dynamics in Rat Heart Mitochondria under ISO-Induced Injury.

Mitochondria are dynamic organelles that produce ATP in the cell and are sensitive to oxidative damage that impairs mitochondrial function in pathological conditions. Mitochondria are involved not only in a healthy heart but also in the development of heart disease. Therefore, attempts should be made to enhance the body's defense response against oxidative stress with the help of various antioxidants in order to decrease mitochondrial damage and reduce mitochondrial dysfunction. Mitochondrial fission and fusion play an important role in the quality control and maintenance of mitochondria. The ketocarotenoid astaxanthin (AX) is an antioxidant able to maintain mitochondrial integrity and prevent oxidative stress. In the present study, we investigated the effect of the protective effect of AX on the functioning of rat heart mitochondria (RHM). Changes in the content of proteins responsible for mitochondrial dynamics, prohibitin 2 (PHB2) as a protein that performs the function of quality control of mitochondrial proteins and participates in the stabilization of mitophagy, and changes in the content of cardiolipin (CL) in rat heart mitochondria after isoproterenol (ISO)-induced damage were examined. AX improved the respiratory control index (RCI), enhanced mitochondrial fusion, and inhibited mitochondrial fission in RHM after ISO injury. Rat heart mitochondria (RHM) were more susceptible to Ca2+-induced mitochondrial permeability pore (mPTP) opening after ISO injection, while AX abolished the effect of ISO. AX is able to perform a protective function in mitochondria, improving their efficiency. Therefore, AX can be considered an important ingredient in the diet for the prevention of cardiovascular disease. Therefore, AX can be examined as an important component of the diet for the prevention of heart disease.

Prohibitin Links Cell Cycle, Motility and Invasion in Prostate Cancer Cells.

Prohibitin (PHB) is a tumour suppressor gene with several different molecular activities. PHB overexpression leads to G1/S-phase cell cycle arrest, and PHB represses the androgen receptor (AR) in prostate cancer cells. PHB interacts with and represses members of the E2F family in a manner that may also be AR-linked, therefore making the AR:PHB:E2F interaction axis highly complex. PHB siRNA increased the growth and metastatic potential of LNCaP mouse xenografts in vivo. Conversely, PHB ectopic cDNA overexpression affected several hundred genes in LNCaP cells. Furthermore, gene ontology analysis showed that in addition to cell cycle regulation, several members of the WNT family were significantly downregulated (WNT7B, WNT9A and WNT10B), as well as pathways for cell adhesion. Online GEO data studies showed PHB expression to be decreased in clinical cases of metastatic prostate cancer, and to be correlated with higher WNT expression in metastasis. PHB overexpression reduced prostate cancer cell migration and motility in wound-healing assays, reduced cell invasion through a Matrigel layer and reduced cellular attachment. In LNCaP cells, WNT7B, WNT9A and WNT10B expression were also upregulated by androgen treatment and downregulated by androgen antagonism, indicating a role for AR in the control of these WNT genes. However, these WNTs were strongly cell cycle regulated. E2F1 cDNA ectopic expression and PHB siRNA (both cell cycle promoting effects) increased WNT7B, WNT9A and WNT10B expression, and these genes were also upregulated as cells were released from G1 to S phase synchronisation, indicating further cell cycle regulation. Therefore, the repressive effects of PHB may inhibit AR, E2F and WNT expression and its loss may increase metastatic potential in human prostate cancer.

PHB promotes bladder cancer cell epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway

As a member of PHB (prohibitin1) family, PHB plays important roles in many cancers, but its property in bladder carcinoma aggressiveness is unknown. This research was to explore the function and potential mechanism of PHB in bladder carcinoma in vivo and in vitro. The invasive abilities of cancer cell were determined by transwell and wound-healing assays. The function of PHB was confirmed by gene knockdown and overexpression methods. Further in vivo confirmation was performed in a nude mouse model with lung metastasis. The relationship of PHB and β-catenin was confirmed by immunoprecipitation and immunofluorescence staining assays. The protein expression of epithelial-mescenchymal transition (EMT) and Wnt/β-catenin signaling pathway was tested by immunofluorescence staining and western blotting assay. The depletion of PHB prevented bladder cancer cell invasiveness and inhibited EMT. Contrarily，the abilities of bladder carcinoma cells migration and invasion in vitro as well as metastasis in vivo were enhanced when the PHB overexpressed unnormally. Importantly, the β-catenin was identified to be bound by PHB and β-catenin knockdown reduced the cancer cell migration, invasion and EMT in PHB overexpressing cells. In addition, PHB stabilized β-catenin by inhibiting its ubiqutin-mediated degradation thus leading to increased Wnt/β-catenin signaling. These observations indicate that PHB could promote bladder cancer aggressiveness by binding with β-catenin to prevent the degradation of β-catenin and the localized invasive bladder cancer patients with PHB overexpression should take more aggressive postsurgical adjuvant anticancer therapies.

Targeted and functional genomics approaches to the mechanism of action of lagunamide D, a mitochondrial cytotoxin from marine cyanobacteria

Lagunamide D, a cyanobacterial cyclodepsipeptide, exhibits potent antiproliferative activity against HCT116 colorectal cancer cells (IC50 5.1 nM), which were used to probe the mechanism of action. Measurements of metabolic activity, mitochondrial membrane potential, caspase 3/7 activity and cell viability indicate the rapid action of lagunamide D on mitochondrial function and downstream cytotoxic effects in HCT116 cells. Lagunamide D preferentially targets the G1 cell cycle population and arrests cells in G2/M phase at high concentration (32 nM). Transcriptomics and subsequent Ingenuity Pathway Analysis identified networks related to mitochondrial functions. Lagunamide D induced mitochondrial network redistribution at 10 nM, suggesting a mechanism shared with the structurally related aurilide family, previously reported to target mitochondrial prohibitin 1 (PHB1). Knockdown and chemical inhibition of ATP1A1 sensitized the cells to lagunamide D, as also known for aurilide B. We interrogated potential mechanisms behind this synergistic effect between lagunamide D and ATP1A1 knockdown by using pharmacological inhibitors and extended the functional analysis to a global level by performing a chemogenomic screen with a siRNA library targeting the human druggable genome, revealing targets that modulate susceptibility to lagunamide D. In addition to mitochondrial targets, the screen revealed hits involved in the ubiquitin/proteasome pathway, suggesting lagunamide D might exert its effects by additionally affecting proteostasis. Our analysis illuminated cellular processes of lagunamide D that can be modulated in parallel to mitochondrial functions. The identification of potential synergistic drug combinations that can alleviate undesirable toxicity may open possibilities to resurrect this class of compounds for anticancer therapy.

Butyrate ameliorates Paneth cell defects, total inflammation, and epithelial cell apoptosis during mitochondrial dysfunction driven by Prohibitin 1-deficiency

Introduction: Prohibitin 1 (PHB1) is a mitochondrial chaperone protein important for maximal activity of the electron transport chain. PHB1 deficiency in intestinal epithelial cells (IECs) or specifically in Paneth cells (PCs) induces epithelial mitochondrial dysfunction, PC defects, gut microbiota dysbiosis, and spontaneous ileitis in mice by 20 weeks-of-age. Dysbiotic gut microbiota has been implicated in inflammatory bowel diseases and is associated with the decreased abundance of short chain fatty acids (SCFAs; acetate, propionate, and butyrate)-producing bacteria. SCFAs play a significant role in reducing intestinal inflammation and enhancing epithelial barrier dysfunction. We tested the hypothesis that luminal SCFA levels were decreased in mice with PHB1 deficiency and that supplementation of SCFAs will ameliorate intestinal inflammation during mitochondrial dysfunction driven by loss of PHB1. Methods: Ileal luminal contents were collected from Phb1fl/fl (control) and Phb1iΔIEC (Phb1-deficient in IECs) mice at 20 weeks of age and SCFAs were measured by gas chromatography-mass spectrometry. Phb1iΔIEC and Phb1fl/fl littermate mice were treated with 20 mM butyrate dissolved in their drinking water from 16 to 20 weeks-of-age and PC defects were measured by antimicrobial peptide expression (Lysozyme, Cryptdin 3, Cryptdin 5, and Ang4) and Lysozyme staining allocation into secretory granules. Ileitis was measured by histological scoring and pro-inflammatory cytokine expression. To test epithelial intrinsic responses, ileal crypt enteroids derived from Phb1iΔIEC and Phb1fl/fl littermate mice were treated with 200 μg/ml healthy ileal stool lysates (from Phb1fl/fl mice) or dysbiotic stool lysates (from Phb1iΔIEC mice) for 16 hours with or without 0.05 mM butyrate, 0.5 mM propionate, 0.5 mM acetate, alone or in combination. Enteroid viability and apoptosis were measured by morphological change and cleaved Caspase 3 expression. Results: Butyrate was significantly decreased in ileal stool of Phb1iΔIEC mice. Butyrate supplementation in Phb1iΔIEC mice protected against ileitis and PC defects. Dysbiotic stool lysate decreased viability and increased apoptosis in Phb1-deficient enteroids that were prevented by butyrate supplementation. Conclusion: Mitochondrial health in IECs cells is crucial for healthy gut microbiota composition and for epithelial defense when the microbiome becomes dysbiotic. Butyrate rescues IECs from insults caused by dysbiotic gut microbiota during mitochondrial dysfunction driven by PHB1 deletion. Funding: APS Postdoctoral Fellowship This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL

AbstractDysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5′ untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation–related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

Essential Protein PHB2 and Its Regulatory Mechanisms in Cancer.

Prohibitins (PHBs) are a highly conserved class of proteins and have an essential role in transcription, epigenetic regulation, nuclear signaling, mitochondrial structural integrity, cell division, and cellular membrane metabolism. Prohibitins form a heterodimeric complex, consisting of two proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2). They have been discovered to have crucial roles in regulating cancer and other metabolic diseases, functioning both together and independently. As there have been many previously published reviews on PHB1, this review focuses on the lesser studied prohibitin, PHB2. The role of PHB2 in cancer is controversial. In most human cancers, overexpressed PHB2 enhances tumor progression, while in some cancers, it suppresses tumor progression. In this review, we focus on (1) the history, family, and structure of prohibitins, (2) the essential location-dependent functions of PHB2, (3) dysfunction in cancer, and (4) the promising modulators to target PHB2. At the end, we discuss future directions and the clinical significance of this common essential gene in cancer.

Identification of Heterophilic Epitopes of H1N1 Influenza Virus Hemagglutinin

Our previous studies found that the H1-50 monoclonal antibody (mAb) of influenza A virus hemagglutinin (HA) cross-reacted with pancreatic tissue and islet β-cells, and further studies showed that H1-50 mAb binds to prohibitin (PHB) protein of islet β-cells. These suggest that there are heterophilic epitopes between influenza virus HA and pancreatic tissue, which may be involved in the pathogenesis of type 1 diabetes. To further investigate these heterophilic epitopes, we screened binding epitopes of H1-50 mAb using a phage 12-peptide library. DNA sequencing and comparative analysis were performed on specific positive phage clones, and the sequence of 12-peptide binding to H1-50 mAb was obtained. The binding epitopes of H1-50 mAb in influenza virus HA were determined by sequence analysis and experimental verification, and their distribution within the three-dimensional structure was assessed by PyMOL. The results showed that H1-50 mAb specifically binds to polypeptides (306-SLPFQNIHPITIGK-319) of influenza A virus HA, located in the stem of the HA protein. However, there is no specific binding sequence between H1-50 mAb and the PHB protein of islet β-cells in the primary structure, and we speculate that the binding of H1-50 mAb to islet β-cells may depend on the spatial conformation. The identification of the heterophilic epitopes of H1N1 influenza virus hemagglutinin provides a new perspective on type 1 diabetes that may be caused by influenza virus infection, which may contribute to the prevention and control of influenza.

RNA interference of PHB1 enhances virulence of Vip3Aa to Sf9 cells and Spodoptera frugiperda larvae.

In our previous work, we demonstrated that prohibitin 2 (PHB2) on the membrane of Sf9 cells was a receptor for Vip3Aa, and PHB2 in mitochondria contributed to the mitochondrial stability to reduce Vip3Aa toxicity. Prohibitin 1 (PHB1), another prohibitin family member, forms heterodimers with PHB2 to maintain the structure and stability of mitochondria. To explore whether PHB1 impacts the action process of Vip3Aa, we examined the correlation between PHB1 and Vip3Aa virulence. We revealed that PHB1 did not colocalize with Vip3Aa in Sf9 cells. The pulldown and CoIP experiments confirmed that PHB1 interacted with neither Vip3Aa nor scavenger receptor-C (another Vip3Aa receptor). Downregulating phb1 expression in Sf9 cells did not affect the internalization of Vip3Aa but increased Vip3Aa toxicity. Further exploration revealed that the decrease of phb1 expression affected mitochondrial function, leading to increased ROS levels and mitochondrial membrane permeability and decreased mitochondrial membrane potential. The increase of mitochondrial cytochrome c release, caspase-3 activity and genomic DNA fragmentation implied that the apoptotic process was also affected. Finally, we applied RNAi to inhibit phb1 expression in Spodoptera frugiperda larvae. As a result, it significantly increased Vip3Aa virulence. We found that PHB1 was not a receptor for Vip3Aa but played an essential role in mitochondria. The downregulation of phb1 expression in Sf9 cells caused instability of mitochondria, and the cells were more prone to apoptosis when challenged with Vip3Aa. The combined use of Vip3Aa and phb1 RNAi showed a synergistic effect against S. frugiperda larvae. © 2023 Society of Chemical Industry.

BUTYRATE AMELIORATES PANETH CELL DEFECTS, TOTAL INFLAMMATION, AND EPITHELIAL CELL APOPTOSIS DURING MITOCHONDRIAL DYSFUNCTION DRIVEN BY PROHIBITIN 1-DEFICIENCY

Prohibitin 1 (PHB1) is a mitochondrial chaperone protein important for maximal activity of the electron transport chain. PHB1 deficiency in intestinal epithelial cells (IECs) or specifically in Paneth cells (PCs) induces epithelial mitochondrial dysfunction, PC defects, gut microbiota dysbiosis, and spontaneous ileitis in mice by 20 weeks-of-age. Dysbiotic gut microbiota has been implicated in inflammatory bowel diseases and is associated with the decreased abundance of short chain fatty acids (SCFAs; acetate, propionate, and butyrate)-producing bacteria. SCFAs play a significant role in reducing intestinal inflammation and epithelial barrier dysfunction. We tested the hypothesis that luminal SCFA levels were decreased in mice with PHB1 deficiency and that supplementation of SCFAs will ameliorate intestinal inflammation during mitochondrial dysfunction driven by loss of PHB1.

Decreasing expression of Prohibitin-2 lowers the oncogenicity of renal cell carcinoma cells by suppressing eIF4E-mediated oncogene translation via MNK inhibition

Prohibitin-2 (PHB2) serves as a key signalling protein that is connected with diverse cellular functions. PHB2 overexpression frequently occurs in cancers and is closely related to tumorigenesis. So far, the connection between PHB2 and renal cell carcinoma (RCC) has not been discussed yet. The purpose of this study was to explore the expression and biological function of PHB2 in RCC and to uncover the underlying mechanisms. High level of PHB2 was found in RCC tissues, and this overexpression was linked to a worse overall survival rate for RCC patients. In RCC cell, the lowering of PHB2 generated tumour-inhibiting effects in RCC cells such as proliferation retardation, cell cycle arrest, suppression of the capacity for metastasis, and chemosensitivity enhancement. Mechanistically, PHB2 mediated the activation of eukaryotic initiation factor 4E (eIF4E) and the translation of oncogenic proteins via the regulation of MNK. The inhibition of MNK diminished the effects of PHB2 on eIF4E-medited oncogene translation. The overexpression of eIF4E reversed PHB2-reduction-evoked tumour-inhibiting effects. Moreover, RCC cells with decreasing PHB2 exhibited a weakened ability to form xenografts in vivo. In conclusion, these findings show that PHB2 is pivotal for RCC progression and suggest that inhibiting MNK/eIF4E by decreasing PHB2 is a potential pathway for the treatment of RCC.

Prohibitin mediates the cellular invasion of spring viremia of the carp virus

Spring viremia of carp virus (SVCV) is strongly contagious and pathogenic to common carp and cyprinoid species. However, knowledge of how SVCV enters host cells is still inadequate. In this study, mass spectrometry (MS) was incorporated with tandem affinity purification (TAP) to identify host proteins that interact with SVCV glycoprotein, the main attachment protein of SVCV. Specifically, prohibitin (PHB) received the utmost attention from all the candidate proteins, and its interaction with the SVCV-G protein was confirmed by immunoprecipitation and immunofluorescence assays. Treatment with PHB-specific inhibitors or knockdown of the expression of PHB by siRNAs resulted in a marked reduction in binding and entry of SVCV on host cells, while overexpression of PHB increased SVCV attachment and invasion. Furthermore, binding of SVCV to ZF4 and FHM cells was inhibited by pre-incubating the virus with recombinant PHB protein (rPHB) or blocking the cell surface PHB with its polyclonal antibodies. In addition, overexpression of PHB on SVCV-nonpermissive Grouper spleen cells (GSs) conferred susceptibility to SVCV infection. In vivo, treatment of rPHB could significantly inhibit SVCV propagation within zebrafish and benefit the survival rate of SVCV-infected zebrafish. These results demonstrate that PHB plays a crucial role in both the attachment and entry stages of SVCV infection.