Pro-opiomelanocortin (POMC) neurons function as key regulators of metabolism and physiology by releasing prohormone-derived neuropeptides with distinct biological activities. However, our understanding of early events in prohormone maturation in the ER remains incomplete. Highlighting the significance of this gap in knowledge, a single POMC cysteine-to-phenylalanine mutation at position 28 (POMC-C28F) is defective for ER processing and causes early onset obesity in a dominant-negative manner in humans through an unclear mechanism. Here, we report a pathologically important role of Sel1L-Hrd1, the protein complex of ER-associated degradation (ERAD), within POMC neurons. Mice with POMC neuron–specific Sel1L deficiency developed age-associated obesity due, at least in part, to the ER retention of POMC that led to hyperphagia. The Sel1L-Hrd1 complex targets a fraction of nascent POMC molecules for ubiquitination and proteasomal degradation, preventing accumulation of misfolded and aggregated POMC, thereby ensuring that another fraction of POMC can undergo normal posttranslational processing and trafficking for secretion. Moreover, we found that the disease-associated POMC-C28F mutant evades ERAD and becomes aggregated due to the presence of a highly reactive unpaired cysteine thiol at position 50. Thus, this study not only identifies ERAD as an important mechanism regulating POMC maturation within the ER, but also provides insights into the pathogenesis of monogenic obesity associated with defective prohormone folding.
Geun Hyang Kim, Guojun Shi, Diane R.M. Somlo, Leena Haataja, Soobin Song, Qiaoming Long, Eduardo A. Nillni, Malcolm J. Low, Peter Arvan, Martin G. Myers Jr., Ling Qi
Infection by Staphylococcus aureus strain USA300 causes tissue injury, multiorgan failure, and high mortality. However, the mechanisms by which the bacteria adhere to, then stabilize on, mucosal surfaces before causing injury remain unclear. We addressed these issues through the first real-time determinations of USA300-alveolar interactions in live lungs. We found that within minutes, inhaled USA300 established stable, self-associated microaggregates in niches at curved, but not at flat, regions of the alveolar wall. The microaggregates released α-hemolysin toxin, causing localized alveolar injury, as indicated by epithelial dye loss, mitochondrial depolarization, and cytosolic Ca2+ increase. Spread of cytosolic Ca2+ through intercellular gap junctions to adjoining, uninfected alveoli caused pulmonary edema. Systemic pretreatment with vancomycin, a USA300-cidal antibiotic, failed to protect mice infected with inhaled WT USA300. However, vancomycin pretreatment markedly abrogated mortality in mice infected with mutant USA300 that lacked the aggregation-promoting factor PhnD. We interpret USA300-induced mortality as having resulted from rapid bacterial aggregation in alveolar niches. These findings indicate, for the first time to our knowledge, that alveolar microanatomy is critical in promoting the aggregation and, hence, in causing USA300-induced alveolar injury. We propose that in addition to antibiotics, strategies for bacterial disaggregation may constitute novel therapy against USA300-induced lung injury.
Jaime L. Hook, Mohammad N. Islam, Dane Parker, Alice S. Prince, Sunita Bhattacharya, Jahar Bhattacharya
Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12–/–) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor–mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12–/– mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12–/– hosts was sufficient to correct the neutrophil migration defect in F12–/– mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.
Evi X. Stavrou, Chao Fang, Kara L. Bane, Andy T. Long, Clément Naudin, Erdem Kucukal, Agharnan Gandhi, Adina Brett-Morris, Michele M. Mumaw, Sudeh Izadmehr, Alona Merkulova, Cindy C. Reynolds, Omar Alhalabi, Lalitha Nayak, Wen-Mei Yu, Cheng-Kui Qu, Howard J. Meyerson, George R. Dubyak, Umut A. Gurkan, Marvin T. Nieman, Anirban Sen Gupta, Thomas Renné, Alvin H. Schmaier
The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a significant percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase USP9X as an FBW7 interactor. USP9X antagonised FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a novel tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations.
Omar M. Khan, Joana Carvalho, Bradley Spencer-Dene, Richard Mitter, David Frith, Ambrosius P. Snijders, Stephen A. Wood, Axel Behrens
Islet amyloidosis is characterized by the aberrant accumulation of islet amyloid polypeptide (IAPP) in pancreatic islets, resulting in β cell toxicity, which exacerbates type 2 diabetes and islet transplant failure. It is not fully clear how IAPP induces cellular stress or how IAPP-induced toxicity can be prevented or treated. We recently defined the properties of toxic IAPP species. Here, we have identified a receptor-mediated mechanism of islet amyloidosis–induced proteotoxicity. In human diabetic pancreas and in cellular and mouse models of islet amyloidosis, increased expression of the receptor for advanced glycation endproducts (RAGE) correlated with human IAPP–induced (h-IAPP–induced) β cell and islet inflammation, toxicity, and apoptosis. RAGE selectively bound toxic intermediates, but not nontoxic forms of h-IAPP, including amyloid fibrils. The isolated extracellular ligand–binding domains of soluble RAGE (sRAGE) blocked both h-IAPP toxicity and amyloid formation. Inhibition of the interaction between h-IAPP and RAGE by sRAGE, RAGE-blocking antibodies, or genetic RAGE deletion protected pancreatic islets, β cells, and smooth muscle cells from h-IAPP–induced inflammation and metabolic dysfunction. sRAGE-treated h-IAPP Tg mice were protected from amyloid deposition, loss of β cell area, β cell inflammation, stress, apoptosis, and glucose intolerance. These findings establish RAGE as a mediator of IAPP-induced toxicity and suggest that targeting the IAPP/RAGE axis is a potential strategy to mitigate this source of β cell dysfunction in metabolic disease.
Andisheh Abedini, Ping Cao, Annette Plesner, Jinghua Zhang, Meilun He, Julia Derk, Sachi A. Patil, Rosa Rosario, Jacqueline Lonier, Fei Song, Hyunwook Koh, Huilin Li, Daniel P. Raleigh, Ann Marie Schmidt
HIF-1α, one of the most extensively studied oncogenes, is activated by a variety of microenvironmental factors. The resulting biological effects are thought to depend on its transcriptional activity. The RNAse enzyme Dicer is frequently downregulated in human cancers, which has been functionally linked to enhanced metastatic properties; however, current knowledge of the upstream mechanisms regulating Dicer is limited. In the present study, we identified Dicer as a HIF-1α–interacting protein in multiple types of cancer cell lines and different human tumors. HIF-1α downregulated Dicer expression by facilitating its ubiquitination by the E3 ligase Parkin, thereby enhancing autophagy-mediated degradation of Dicer, which further suppressed the maturation of known tumor suppressors, such as the microRNA let-7 and microRNA-200b. Consequently, expression of HIF-1α facilitated epithelial-mesenchymal transition (EMT) and metastasis in tumor-bearing mice. Thus, this study uncovered a connection between oncogenic HIF-1α and the tumor-suppressive Dicer. This function of HIF-1α is transcription independent and occurs through previously unrecognized protein interaction–mediated ubiquitination and autophagic proteolysis.
Hui-Huang Lai, Jie-Ning Li, Ming-Yang Wang, Hsin-Yi Huang, Carlo M. Croce, Hui-Lung Sun, Yu-Jhen Lyu, Jui-Wen Kang, Ching-Feng Chiu, Mien-Chie Hung, Hiroshi I. Suzuki, Pai-Sheng Chen
The molecular mechanism by which cancer-associated fibroblasts (CAFs) confer chemoresistance in ovarian cancer is poorly understood. The purpose of the present study was to evaluate the roles of CAFs in modulating tumor vasculature, chemoresistance, and disease progression. Here, we found that CAFs upregulated the lipoma-preferred partner (LPP) gene in microvascular endothelial cells (MECs) and that LPP expression levels in intratumoral MECs correlated with survival and chemoresistance in patients with ovarian cancer. Mechanistically, LPP increased focal adhesion and stress fiber formation to promote endothelial cell motility and permeability. siRNA-mediated LPP silencing in ovarian tumor–bearing mice improved paclitaxel delivery to cancer cells by decreasing intratumoral microvessel leakiness. Further studies showed that CAFs regulate endothelial LPP via a calcium-dependent signaling pathway involving microfibrillar-associated protein 5 (MFAP5), focal adhesion kinase (FAK), ERK, and LPP. Thus, our findings suggest that targeting endothelial LPP enhances the efficacy of chemotherapy in ovarian cancer. Our data highlight the importance of CAF–endothelial cell crosstalk signaling in cancer chemoresistance and demonstrate the improved efficacy of using LPP-targeting siRNA in combination with cytotoxic drugs.
Cecilia S. Leung, Tsz-Lun Yeung, Kay-Pong Yip, Kwong-Kwok Wong, Samuel Y. Ho, Lingegowda S. Mangala, Anil K. Sood, Gabriel Lopez-Berestein, Jianting Sheng, Stephen T.C. Wong, Michael J. Birrer, Samuel C. Mok
DNA double-strand breaks (DSBs) are mainly repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) pathways. Here, we showed that myeloid cell leukemia sequence 1 (Mcl-1) acts as a functional switch in selecting between HR and NHEJ pathways. Mcl-1 was cell cycle–regulated during HR, with its expression peaking in S/G2 phase. While endogenous Mcl-1 depletion reduced HR and enhanced NHEJ, Mcl-1 overexpression resulted in a net increase in HR over NHEJ. Mcl-1 directly interacted with the dimeric Ku protein complex via its Bcl-2 homology 1 and 3 (BH1 and BH3) domains, which are required for Mcl-1 to inhibit Ku-mediated NHEJ. Mcl-1 also promoted DNA resection mediated by the Mre11 complex and HR-dependent DSB repair. Using the Mcl-1 BH1 domain as a docking site, we identified a small molecule, MI-223, that directly bound to BH1 and blocked Mcl-1–stimulated HR DNA repair, leading to sensitization of cancer cells to hydroxyurea- or olaparib-induced DNA replication stress. Combined treatment with MI-223 and hydroxyurea or olaparib exhibited a strong synergy against lung cancer in vivo. This mechanism-driven combination of agents provides a highly attractive therapeutic strategy to improve lung cancer outcomes.
Guo Chen, Andrew T. Magis, Ke Xu, Dongkyoo Park, David S. Yu, Taofeek K. Owonikoko, Gabriel L. Sica, Sarah W. Satola, Suresh S. Ramalingam, Walter J. Curran, Paul W. Doetsch, Xingming Deng
SHARPIN, an adaptor for the linear ubiquitin chain assembly complex (LUBAC), plays important roles in NF-κB signaling and inflammation. Here, we have demonstrated a LUBAC-independent role for SHARPIN in regulating melanoma growth. We observed that SHARPIN interacted with PRMT5, a type II protein arginine methyltransferase, and increased its multiprotein complex and methyltransferase activity. Activated PRMT5 controlled the expression of the transcription factors SOX10 and MITF by SHARPIN-dependent arginine dimethylation and inhibition of the transcriptional corepressor SKI. Activation of PRMT5 by SHARPIN counteracted PRMT5 inhibition by methylthioadenosine, a substrate of methylthioadenosine phosphorylase, which is codeleted with cyclin-dependent kinase inhibitor 2A (CDKN2A) in approximately 15% of human cancers. Collectively, we identified a LUBAC-independent role for SHARPIN in enhancing PRMT5 activity that contributes to melanomagenesis through the SKI/SOX10 regulatory axis.
Hironari Tamiya, Hyungsoo Kim, Oleksiy Klymenko, Heejung Kim, Yongmei Feng, Tongwu Zhang, Ji Yun Han, Ayako Murao, Scott J. Snipas, Lucia Jilaveanu, Kevin Brown, Harriet Kluger, Hao Zhang, Kazuhiro Iwai, Ze’ev A. Ronai
The tumor suppressor protein retinoblastoma (RB) is mechanistically linked to suppression of transcription factor E2F1-mediated cell cycle regulation. For multiple tumor types, loss of RB function is associated with poor clinical outcome. RB action is abrogated either by direct depletion or through inactivation of RB function; however, the basis for this selectivity is unknown. Here, analysis of tumor samples and cell-free DNA from patients with advanced prostate cancer showed that direct RB loss was the preferred pathway of disruption in human disease. While RB loss was associated with lethal disease, RB-deficient tumors had no proliferative advantage and exhibited downstream effects distinct from cell cycle control. Mechanistically, RB loss led to E2F1 cistrome expansion and different binding specificity, alterations distinct from those observed after functional RB inactivation. Additionally, identification of protumorigenic transcriptional networks specific to RB loss that were validated in clinical samples demonstrated the ability of RB loss to differentially reprogram E2F1 in human cancers. Together, these findings not only identify tumor-suppressive functions of RB that are distinct from cell cycle control, but also demonstrate that the molecular consequence of RB loss is distinct from RB inactivation. Thus, these studies provide insight into how RB loss promotes disease progression, and identify new nodes for therapeutic intervention.
Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Mateus Crespo, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A. Rubin, Myles Brown, Karen E. Knudsen
No posts were found with this tag.