Dual-specificity phosphatase 1 interacts with prohibitin 2 to improve mitochondrial quality control against type-3 cardiorenal syndrome.

Type-3 cardiorenal syndrome (CRS-3) is acute kidney injury followed by cardiac injury/dysfunction. Mitochondrial injury may impair myocardial function during CRS-3. Since dual-specificity phosphatase 1 (DUSP1) and prohibitin 2 (PHB2) both promote cardiac mitochondrial quality control, we assessed whether these proteins were dysregulated during CRS-3-related cardiac depression. We found that DUSP1 was downregulated in heart tissues from a mouse model of CRS-3. DUSP1 transgenic (DUSP1Tg) mice were protected from CRS-3-induced myocardial damage, as evidenced by their improved heart function and myocardial structure. CRS-3 induced the inflammatory response, oxidative stress and mitochondrial dysfunction in wild-type hearts, but not in DUSP1Tg hearts. DUSP1 overexpression normalized cardiac mitochondrial quality control during CRS-3 by suppressing mitochondrial fission, restoring mitochondrial fusion, re-activating mitophagy and augmenting mitochondrial biogenesis. We found that DUSP1 sustained cardiac mitochondrial quality control by binding directly to PHB2 and maintaining PHB2 phosphorylation, while CRS-3 disrupted this physiological interaction. Transgenic knock-in mice carrying the Phb2S91D variant were less susceptible to cardiac depression upon CRS-3, due to a reduced inflammatory response, suppressed oxidative stress and improved mitochondrial quality control in their heart tissues. Thus, CRS-3-induced myocardial dysfunction can be attributed to reduced DUSP1 expression and disrupted DUSP1/PHB2 binding, leading to defective cardiac mitochondrial quality control.

Prohibitin1 facilitates viral replication by impairing the RIG-I-like receptor signaling pathway.

IMPORTANCE: Type I interferon (IFN-I), produced by the innate immune system, plays an essential role in host antiviral responses. Proper regulation of IFN-I production is required for the host to balance immune responses and prevent superfluous inflammation. IFN regulatory factor 3 (IRF3) and subsequent sensors are activated by RNA virus infection to induce IFN-I production. Therefore, proper regulation of IRF3 serves as an important way to control innate immunity and viral replication. Here, we first identified Prohibitin1 (PHB1) as a negative regulator of host IFN-I innate immune responses. Mechanistically, PHB1 inhibited the nucleus import of IRF3 by impairing its binding with importin subunit alpha-1 and importin subunit alpha-5. Our study demonstrates the mechanism by which PHB1 facilitates the replication of multiple RNA viruses and provides insights into the negative regulation of host immune responses.

Prohibitin1 maintains mitochondrial quality in isoproterenol-induced cardiac hypertrophy in H9C2 cells.

BACKGROUND INFORMATION: Various types of stress initially induce a state of cardiac hypertrophy (CH) in the heart. But, persistent escalation of cardiac stress leads to progression from an adaptive physiological to a maladaptive pathological state. So, elucidating molecular mechanisms that can attenuate CH is imperative in developing cardiac therapies. Previously, we showed that Prohibitin1 (PHB1) has a protective role in CH-induced oxidative stress. Nevertheless, it is unclear how PHB1, a mitochondrial protein, has a protective role in CH. Therefore, we hypothesized that PHB1 maintains mitochondrial quality in CH. To test this hypothesis, we used Isoproterenol (ISO) to induce CH in H9C2 cells overexpressing PHB1 and elucidated mitochondrial quality control pathways. RESULTS: We found that overexpressing PHB1 attenuates ISO-induced CH and restores mitochondrial morphology in H9C2 cells. In addition, PHB1 blocks the pro-hypertrophic IGF1R/AKT pathway and restores the mitochondrial membrane polarization in ISO-treated cells. We observed that overexpressing PHB1 promotes mitochondrial biogenesis, improves mitochondrial respiratory capacity, and triggers mitophagy. CONCLUSION: We conclude that PHB1 maintains mitochondrial quality in ISO-induced CH in H9C2 cells. SIGNIFICANCE: Based on our results, we suggest that small molecules that induce PHB1 in cardiac cells may prove beneficial in developing cardiac therapies.

GALNT14-mediated O-glycosylation on PHB2 serine-161 enhances cell growth, migration and drug resistance by activating IGF1R cascade in hepatoma cells.

The single nucleotide polymorphism (SNP) rs9679162 located on GALNT14 gene predicts therapeutic outcomes in patients with intermediate and advanced hepatocellular carcinoma (HCC), but the molecular mechanism remains unclear. Here, the associations between SNP genotypes, GALNT14 expression, and downstream molecular events were determined. A higher GALNT14 cancerous/noncancerous ratio was associated with the rs9679162-GG genotype, leading to an unfavorable postoperative prognosis. A novel exon-6-skipped GALNT14 mRNA variant was identified in patients carrying the rs9679162-TT genotype, which was associated with lower GALNT14 expression and favorable prognosis. Cell-based experiments showed that elevated levels of GALNT14 promoted HCC growth, migration, and resistance to anticancer drugs. Using a comparative lectin-capture glycoproteomic approach, PHB2 was identified as a substrate for GALNT14-mediated O-glycosylation. Site-directed mutagenesis experiments revealed that serine-161 (Ser161) was the O-glycosylation site. Further analysis showed that O-glycosylation of PHB2-Ser161 was required for the GALNT14-mediated growth-promoting phenotype. O-glycosylation of PHB2 was positively correlated with GALNT14 expression in HCC, resulting in increased interaction between PHB2 and IGFBP6, which in turn led to the activation of IGF1R-mediated signaling. In conclusion, the GALNT14-rs9679162 genotype was associated with differential expression levels of GALNT14 and the generation of a novel exon-6-skipped GALNT14 mRNA variant, which was associated with a favorable prognosis in HCC. The GALNT14/PHB2/IGF1R cascade modulated the growth, migration, and anticancer drug resistance of HCC cells, thereby opening the possibility of identifying new therapeutic targets against HCC.

The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy.

The autophagic clearance of mitochondria has been defined as mitophagy, which is triggered by mitochondrial damage and serves as a major pathway for mitochondrial homeostasis and cellular quality control. PINK1 and Parkin-mediated mitophagy is the most extensively studied form of mitophagy, which has been linked to the pathogenesis of neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The current paradigm of this particular mitophagy pathway is that the ubiquitination of the outer mitochondrial membrane is the key step to enable the recognition of damaged mitochondria by the core autophagic component autophagosome. However, whether the inner mitochondrial membrane (IMM) is ubiquitinated by Parkin and its contribution to sufficient mitophagy remain unclear. Here, using molecular, cellular, and biochemical approaches, we report that prohibitin 2 (PHB2), an essential IMM receptor for mitophagy, is ubiquitinated by Parkin and thereby gains higher affinity to the autophagosome during mitophagy. Our findings suggest that Parkin directly binds to PHB2 through its RING1 domain and promotes K11- and K33-linked ubiquitination on K142/K200 sites of PHB2, thereby enhancing the interaction between PHB2 and MAP1LC3B/LC3B. Interestingly and importantly, our study allows us to propose a novel model in which IMM protein PHB2 serves as both a receptor and a ubiquitin-mediated base for autophagosome recruitment to ensure efficient mitophagy.

Prohibitin 1 regulates mtDNA release and downstream inflammatory responses.

Exposure of mitochondrial DNA (mtDNA) to the cytosol activates innate immune responses. But the mechanisms by which mtDNA crosses the inner mitochondrial membrane are unknown. Here, we found that the inner mitochondrial membrane protein prohibitin 1 (PHB1) plays a critical role in mtDNA release by regulating permeability across the mitochondrial inner membrane. Loss of PHB1 results in alterations in mitochondrial integrity and function. PHB1-deficient macrophages, serum from myeloid-specific PHB1 KO (Phb1MyeKO) mice, and peripheral blood mononuclear cells from neonatal sepsis patients show increased interleukin-1ß (IL-1ß) levels. PHB1 KO mice are also intolerant of lipopolysaccharide shock. Phb1-depleted macrophages show increased cytoplasmic release of mtDNA and inflammatory responses. This process is suppressed by cyclosporine A and VBIT-4, which inhibit the mitochondrial permeability transition pore (mPTP) and VDAC oligomerization. Inflammatory stresses downregulate PHB1 expression levels in macrophages. Under normal physiological conditions, the inner mitochondrial membrane proteins, AFG3L2 and SPG7, are tethered to PHB1 to inhibit mPTP opening. Downregulation of PHB1 results in enhanced interaction between AFG3L2 and SPG7, mPTP opening, mtDNA release, and downstream inflammatory responses.

Protein disulfide isomerase blocks the interaction of LC3II-PHB2 and promotes mTOR signaling to regulate autophagy and radio/chemo-sensitivity.

Protein disulfide isomerase (PDI) is an endoplasmic reticulum (ER) enzyme that mediates the formation of disulfide bonds, and is also a therapeutic target for cancer treatment. Our previous studies found that PDI mediates apoptotic signaling by inducing mitochondrial dysfunction. Considering that mitochondrial dysfunction is a major contributor to autophagy, how PDI regulates autophagy remains unclear. Here, we provide evidence that high expression of PDI in colorectal cancer tumors significantly increases the risk of metastasis and poor prognosis of cancer patients. PDI inhibits radio/chemo-induced cell death by regulating autophagy signaling. Mechanistically, the combination of PDI and GRP78 was enhanced after ER stress, which inhibits the degradation of AKT by GRP78, and eventually activates the mTOR pathway to inhibit autophagy initiation. In parallel, PDI can directly interact with the mitophagy receptor PHB2 in mitochondrial, then competitively blocks the binding of LC3II and PHB2 and inhibits the mitophagy signaling. Collectively, our results identify that PDI can reduce radio/chemo-sensitivity by regulating autophagy, which could be served as a potential target for radio/chemo-therapy.

The Yun/Prohibitin complex regulates adult Drosophila intestinal stem cell proliferation through the transcription factor E2F1.

Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. Here, from an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), we identify a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; our genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. We demonstrate that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, we find that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, our study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation.

MicroRNA-24-3p alleviates cardiac fibrosis by suppressing cardiac fibroblasts mitophagy via downregulating PHB2.

Cardiac fibrosis is a pathological process of multiple cardiovascular diseases, which may lead to heart failure. Studies have shown that microRNAs (miRNAs) play critical roles in regulating mitophagy and cardiac fibrosis. We found that miR-24-3p expression was significantly downregulated in transverse aortic constriction (TAC) mice and cardiac fibroblasts (CFs) treated with Ang Ⅱ. We also found that, apart from improving cardiac structure and function, forced expression of miR-24-3p not only reduced the levels of collagen and α-SMA but also inhibited proliferation and migration of CFs. Next, our research proved that miR-24-3p suppressed the progression of mitophagy, autophagic flux, and the levels of mitophagy-related proteins in cardiac fibrosis models. Further analysis showed that PHB2 was a direct target of miR-24-3p. Finally, experiments showed that the knockdown of PHB2 reversed Ang Ⅱ-induced fibrosis in CFs. The results of our study suggests that increased expression of miR-24-3p contributes to the reduction of cardiac fibrosis and that it might be targeted therapeutically to alleviate cardiac fibrosis.

The Mitochondrial PHB2/OMA1/DELE1 Pathway Cooperates with Endoplasmic Reticulum Stress to Facilitate the Response to Chemotherapeutics in Ovarian Cancer.

Interactions between the mitochondrial inner and outer membranes and between mitochondria and other organelles closely correlates with the sensitivity of ovarian cancer to cisplatin and other chemotherapeutic drugs. However, the underlying mechanism remains unclear. Recently, the mitochondrial protease OMA1, which regulates internal and external signals in mitochondria by cleaving mitochondrial proteins, was shown to be related to tumor progression. Therefore, we evaluated the effect of OMA1 on the response to chemotherapeutics in ovarian cancer cells and the mouse subcutaneous tumor model. We found that OMA1 activation increased ovarian cancer sensitivity to cisplatin in vivo and in vitro. Mechanistically, in ovarian cancer, OMA1 cleaved optic atrophy 1 (OPA1), leading to mitochondrial inner membrane cristae remodeling. Simultaneously, OMA1 induced DELE1 cleavage and its cytoplasmic interaction with EIF2AK1. We also demonstrated that EIF2AK1 cooperated with the ER stress sensor EIF2AK3 to amplify the EIF2S1/ATF4 signal, resulting in the rupture of the mitochondrial outer membrane. Knockdown of OMA1 attenuated these activities and reversed apoptosis. Additionally, we found that OMA1 protease activity was regulated by the prohibitin 2 (PHB2)/stomatin-like protein 2 (STOML2) complex. Collectively, OMA1 coordinates the mitochondrial inner and outer membranes to induce ovarian cancer cell death. Thus, activating OMA1 may be a novel treatment strategy for ovarian cancer.

Preparation and Application of a Chemical Probe for Identifying the Targets of the Marine Cyclic Peptide Kapakahine A.

Marine organisms are a rich source of bioactive secondary metabolites. Although many marine natural products with bioactivities have been isolated, successful elucidation of their mechanisms of action remains limited. In this study, we prepared a probe molecule based on the marine cyclic peptide kapakahine A (1) by introducing a linker with an azide terminal group, which enables the introduction of fluorescent groups for the effective monitoring of subcellular localization, or coupling to affinity beads for the pull-down of target proteins. The results of LC/MS/MS measurements, ProteinPilot analysis, and Western blotting suggest that kapakahine A interacts with the mitochondrial inner membrane proteins PHB1, PHB2, and ANT2, which is consistent with the results of the subcellular localization analysis using a fluorescent probe.

Membrane prohibitin forms a dynamic complex with p56lck to regulate T cell receptor signaling.

Prohibitin is a highly conserved ubiquitously expressed protein involved in several key cellular functions. Targeting of this protein in the membrane by the virulence polysaccharide, Vi, of human typhoid-causing pathogen, Salmonella enterica serovar Typhi (S. Typhi), results in suppression of IL-2 secretion from T cells activated through the T-cell receptor (TCR). However, the mechanism of this suppression remains unclear. Here, using Vi as a probe, we show that membrane prohibitin associates with the src-tyrosine kinase, p56lck (Lck), and actin in human model T cell line, Jurkat. Activation with anti-CD3 antibody brings about dissociation of this complex, which coincides with downstream ERK activation. The trimolecular complex reappears towards culmination of proximal TCR signaling. Engagement of cells with Vi prevents TCR-triggered activation of Lck and ERK by inhibiting dissociation of the former from prohibitin. These findings suggest a regulatory role for membrane prohibitin in Lck activation and TCR signaling.

Fluorizoline Blocks the Interaction between Prohibitin-2 and γ-Glutamylcyclotransferase and Induces p21Waf1/Cip1 Expression in MCF7 Breast Cancer Cells.

Prohibitin-2 (PHB2) is a scaffold protein that has pleiotropic functions, which include interacting with γ-glutamylcyclotransferase (GGCT) in the cytoplasm and repressing the transcriptional activities of the p21Waf1/Cip (p21) gene in the nucleus. The cytotoxic drug fluorizoline binds to PHB1/2 and exerts antiproliferative actions on cancer cells. However, the precise mechanism underlying the antiproliferative effects of fluorizoline is not fully elucidated. In the present study, we first show that fluorizoline induces p21 expression in several human cancer cell lines, including MCF7 breast cancer cells. Treatment of MCF7 cells with fluorizoline suppressed proliferation and prevented cells from entering into the DNA synthesis phase. Knockdown of p21 rescued the suppressed proliferation, indicating that fluorizoline inhibited MCF7 cell growth via the induction of p21. Overexpression of PHB2 in MCF7 cells prevented the induction of p21 expression by fluorizoline and restored the antiproliferative effects and blockade of cell cycle progression. Moreover, treatment of MCF7 cells with fluorizoline inhibited the interaction between endogenous PHB2 and GGCT proteins and reduced the level of nuclear localization of PHB2 proteins. These results indicate that targeting PHB2 with fluorizoline induces the expression of p21 and consequently blocks proliferation of cancer cells. SIGNIFICANCE STATEMENT: This study shows that fluorizoline may be a promising novel anticancer drug candidate that induces p21 expression and blocks cell-cycle progression in human cancer cell lines. In addition, we show that fluorizoline inhibits the interaction between PHB2 and GGCT and reduces the nuclear localization of PHB2 proteins.

The expression of PHB2 in the cochlea: Possible relation to age-related hearing loss.

Age-related hearing loss (ARHL) is the most prevalent sensory deficit in the elderly, but its mechanism remains unclear. Scaffold protein prohibitin 2 (PHB2) has been widely involved in aging and neurodegeneration. However, the role of PHB2 in ARHL is undeciphered to date. To investigate the expression pattern and the role of PHB2 in ARHL, we used C57BL/6 mice and HEI-OC1 cell line as models. In our study, we have found PHB2 exists in the cochlea and is expressed in hair cells, spiral ganglion neurons, and HEI-OC1 cells. In mice with ARHL, mitophagy is reduced and correspondingly the expression level of PHB2 is decreased. Moreover, after H2 O2 treatment the mitophagy is activated and the PHB2 expression is increased. These findings indicate that PHB2 may exert an important role in ARHL through mitophagy. Findings from this study will be helpful for elucidating the mechanism underlying the ARHL and for providing a new target for ARHL treatment.

Prohibitin Inactivation in Adipocytes Results in Reduced Lipid Metabolism and Adaptive Thermogenesis Impairment.

Prohibitin-1 (PHB) is a multifunctional protein previously reported to be important for adipocyte function. PHB is expressed on the surface of adipose cells, where it interacts with a long-chain fatty acid (LCFA) transporter. Here, we show that mice lacking PHB in adipocytes (PHB adipocyte [Ad]-knockout [KO]) have a defect in fat tissue accumulation despite having larger lipid droplets in adipocytes due to reduced lipolysis. Although PHB Ad-KO mice do not display glucose intolerance, they are insulin resistant. We show that PHB Ad-KO mice are lipid intolerant due to a decreased capacity of adipocytes for LCFA uptake. Instead, PHB Ad-KO mice have increased expression of GLUT1 in various tissues and use glucose as a preferred energy source. We demonstrate that PHB Ad-KO mice have defective brown adipose tissue, are intolerant to cold, and display reduced basal energy expenditure. Systemic repercussions of PHB inactivation in adipocytes were observed in both males and females. Consistent with lower cellular mitochondrial content and reduced uncoupling protein 1 protein expression, brown adipocytes lacking PHB display decreased proton leak and switch from aerobic metabolism to glycolysis. Treatment of differentiating brown adipocytes with small molecules targeting PHB suppressed mitochondrial respiration and uncoupling. Our results demonstrate that PHB in adipocytes is essential for normal fatty acid uptake, oxidative metabolism, and adaptive thermogenesis. We conclude that PHB inhibition could be investigated as an approach to altering energy substrate utilization.

Folate receptor α increases chemotherapy resistance through stabilizing MDM2 in cooperation with PHB2 that is overcome by MORAb-202 in gastric cancer.

BACKGROUND: The main function of folate receptor α (FOLRα) has been considered to mediate intracellular folate uptake and induce tumor cell proliferation. Given the broad spectrum of expression among malignant tumors, including gastric cancer (GC) but not in normal tissue, FOLRα represents an attractive target for tumor-selective drug delivery. However, the efficacy of anti-FOLRα monoclonal antibodies (mAbs) has not been proved so far, with the reason for this failure remaining unclear, raising the need for a better understanding of FOLRα function. METHODS: The distribution of FOLRα in GC cells was evaluated by immunohistochemistry. The impacts of FOLRα expression on the survival of GC patients and GC cell lines were examined with the Gene Expression Omnibus database and by siRNA of FOLRα. RNA-sequencing and Microarray analysis was conducted to identify the function of FOLRα. Proteins that interact with FOLRα were identified with shotgun LC-MS/MS. The antitumor efficacy of the anti-FOLRα mAb farletuzumab as well as the antibody-drug conjugate (ADC) consists of the farletuzumab and the tublin-depolymerizing agent eribulin (MORAb-202) was evaluated both in vitro and in vivo. RESULTS: FOLRα was detected both at the cell membrane and in the cytoplasm. Shorter overall survival was associated with FOLRα expression in GC patients, whereas reduction of FOLRα attenuated cell proliferation without inducing cell death in GC cell lines. Transcriptomic and proteomic examinations revealed that the FOLRα-expressing cancer cells possess a mechanism of chemotherapy resistance supported by MDM2, and FOLRα indirectly regulates it through a chaperone protein prohibitin2 (PHB2). Although reduction of FOLRα brought about vulnerability for oxaliplatin by diminishing MDM2 expression, farletuzumab did not suppress the MDM2-mediated chemoresistance and cell proliferation in GC cells. On the other hand, MORAb-202 showed significant antitumor efficacy. CONCLUSIONS: The ADC could be a more reasonable choice than mAb as a targeting agent for the FOLRα-expressing tumor.