The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB3, EccC3, and EccE3 and two copies of the EccD3 protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters.

Chlamydia trachomatis is an obligate intracellular pathogen that resides in a membrane-bound compartment, the inclusion. The bacteria secrete a unique class of proteins, Incs, which insert into the inclusion membrane and modulate the host-bacterium interface. We previously reported that IncE binds specifically to the Sorting Nexin 5 Phox domain (SNX5-PX) and disrupts retromer trafficking. Here, we present the crystal structure of the SNX5-PX:IncE complex, showing IncE bound to a unique and highly conserved hydrophobic groove on SNX5. Mutagenesis of the SNX5-PX:IncE binding surface disrupts a previously unsuspected interaction between SNX5 and the cation-independent mannose-6-phosphate receptor (CI-MPR). Addition of IncE peptide inhibits the interaction of CI-MPR with SNX5. Finally, C. trachomatis infection interferes with the SNX5:CI-MPR interaction, suggesting that IncE and CI-MPR are dependent on the same binding surface on SNX5. Our results provide new insights into retromer assembly and underscore the power of using pathogens to discover disease-related cell biology.DOI: http://dx.doi.org/10.7554/eLife.22709.001

Urothelial cell carcinoma of the bladder (UCCB) is the most common form of bladder cancer and it is estimated that ~15,000 people in the US succumbed to this disease in 2013. Bladder cancer treatment options are limited and research to understand the molecular mechanisms of this disease is needed to design novel therapeutic strategies. Recent studies have shown that microRNAs play pivotal roles in the progression of cancer. MiR-148a has been shown to serve as a tumor suppressor in cancers of the prostate, colon, and liver, but its role in bladder cancer has never been elucidated. Here we show that miR-148a is down-regulated in UCCB cell lines. We demonstrate that overexpression of miR-148a leads to reduced cell viability through an increase in apoptosis rather than an inhibition of proliferation. We additionally show that miR-148a exerts this effect partially by attenuating expression of DNA methyltransferase 1 (DNMT1). Finally, our studies demonstrate that treating cells with both miR-148a and either cisplatin or doxorubicin is either additive or synergistic in causing apoptosis. These data taken together suggest that miR-148a is a tumor suppressor in UCCB and could potentially serve as a novel therapeutic for this malignancy.

Few patients with triple negative breast cancer (TNBC) benefit from immune checkpoint inhibitors with complete and durable remissions being quite rare. Oncogenes can regulate tumor immune infiltration, however whether oncogenes dictate diminished response to immunotherapy and whether these effects are reversible remains poorly understood. Here, we report that TNBCs with elevated MYC expression are resistant to immune checkpoint inhibitor therapy. Using mouse models and patient data, we show that MYC signaling is associated with low tumor cell PD-L1, low overall immune cell infiltration, and low tumor cell MHC-I expression. Restoring interferon signaling in the tumor increases MHC-I expression. By combining a TLR9 agonist and an agonistic antibody against OX40 with anti-PD-L1, mice experience tumor regression and are protected from new TNBC tumor outgrowth. Our findings demonstrate that MYC-dependent immune evasion is reversible and druggable, and when strategically targeted, may improve outcomes for patients treated with immune checkpoint inhibitors.

Treatment options for high grade urothelial cancers are limited and have remained largely unchanged for several decades. Selinexor (KPT-330), a first in class small molecule that inhibits the nuclear export protein XPO1, has shown efficacy as a single agent treatment for numerous different malignancies, but its efficacy in limiting bladder malignancies has not been tested. In this study we assessed selinexor-dependent cytotoxicity in several bladder tumor cells and report that selinexor effectively reduced XPO1 expression and limited cell viability in a dose dependent manner. The decrease in cell viability was due to an induction of apoptosis and cell cycle arrest. These results were recapitulated in in vivo studies where selinexor decreased tumor growth. Tumors treated with selinexor expressed lower levels of XPO1, cyclin A, cyclin B, and CDK2 and increased levels of RB and CDK inhibitor p27, a result that is consistent with growth arrest. Cells expressing wildtype RB, a potent tumor suppressor that promotes growth arrest and apoptosis, were most susceptible to selinexor. Cell fractionation and immunofluorescence studies showed that selinexor treatment increased nuclear RB levels and mechanistic studies revealed that RB ablation curtailed the response to the drug. Conversely, limiting CDK4/6 dependent RB phosphorylation by palbociclib was additive with selinexor in reducing bladder tumor cell viability, confirming that RB activity has a role in the response to XPO1 inhibition. These results provide a rationale for XPO1 inhibition as a novel strategy for the treatment of bladder malignancies.

Dicer expression is frequently altered in cancer and affects a wide array of cellular functions acting as an oncogene or tumor suppressor in varying contexts. It has been shown that Dicer expression is also deregulated in urothelial cell carcinoma of the bladder (UCCB) but the nature of this deregulation differs between reports. The aim of the present study was to gain a better understanding of the role of Dicer in bladder cancer to help determine its contribution to the disease. The results showed that Dicer transcript levels were decreased in UCCB tumor tissues as compared to normal tissues, suggesting that Dicer is a tumor suppressor. However, consistent with previous results, we demonstrated that knockdown of Dicer decreases cell viability and increases the induction of apoptosis, suggesting that Dicer is an oncogene. To resolve this discrepancy, we assessed the effects of decreased Dicer expression on epithelial-to‑mesenchymal transition, migration and invasion. We showed that decreased Dicer levels promoted a mesenchymal phenotype and increased migration. Additionally, the results showed that Dicer protein ablation leads to increased cell invasion, higher levels of matrix metalloproteinase-2, and decreased levels of key miRNAs shown to inhibit invasion. The results of this study suggest that decreased Dicer levels may portend a more malignant phenotype.

No abstract

11The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and 12 many Gram-positive bacteria that mediate a broad range of functions including virulence, 13 conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-14 superfamily protein substrates across the cytoplasmic membrane; however, the architecture and 15 mechanism of translocation has remained elusive. We report the cryo-electron microscopy 16 structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the 17 model organism Mycobacterium smegmatis. The structure reveals two large a-helical membrane 18 pores of sufficient diameter to secrete folded substrates. A complex, asymmetric, multimeric 19 cytoplasmic domain is poised to gate and regulate the pore's function. Our study provides 20 mechanistic insights into the ESX systems and will guide structure-based design of drugs 21 targeting this unique bacterial translocon. 22 23 One Sentence Summary: The structure of the ESX-3 secretion system reveals a pore of 24 sufficient size for the transit of folded substrates and a complex, cytoplasmic regulatory 25 apparatus. 26 65 expression of ESX-3 ( Fig. S2A). Four components of the complex EccB3, EccC3, EccD3 and 66 EccE3 were affinity-purified as a large molecular weight species (Fig. 1B and 1C, Fig. S2B) 67 using a GFP-nanobody and the GFP tag was proteolytically cleaved. 68 The ESX-3 translocon complex was imaged by cryo-EM and reconstructed revealing a 69 dimeric structure which can be divided into four functionally important areas, the transmembrane 70 translocon pore, the cytoplasmic translocon gate, the cytoplasmic motor domain, and the 71