ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.

Rab1b-GBF1-ARF1 secretory pathway axis is required for Birnavirus replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of non-enveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the G olgi c omplex (GC). In this work, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. Analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector G olgi-specific B FA resistance f actor 1 (GBF1), which activates the small GTPase A DP- r ibosylation f actor 1 (ARF1), is required for IBDV replication since inhibiting its activity by treatment with b re f eldin A (BFA) or G olgi c ide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative-mutant T31N over-expression hampered the IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnaviruses-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, being the lack of a transcriptionally active core the main differential feature. This structural trait, among others that resemble the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and have argued the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses. Here, we present original data showing the IBDV-induced GC reorganization and the crosstalk between IBDV and the Rab1b-GBF1-ARF1 mediated intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnaviruses-host cells and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

Discovering and Characterizing of Survivin Dominant Negative Mutants With Stronger Pro-apoptotic Activity on Cancer Cells and CSCs

Survivin as a member of the inhibitor of apoptosis proteins (IAPs) family is undetectable in normal cells, but highly expressed in cancer cells and cancer stem cells (CSCs) which makes it an attractive target in cancer therapy. Survivin dominant negative mutants have been reported as competitive inhibitors of endogenous survivin protein in cancer cells. However, there is a lack of systematic comparative studies on which mutants have stronger effect on promoting apoptosis in cancer cells, which will hinder the development of novel anti-cancer drugs. Here, based on the previous study of survivin and its analysis of the relationship between structure and function, we designed and constructed a series of different amino acid mutants from survivin (TmSm34, TmSm48, TmSm84, TmSm34/48, TmSm34/84, and TmSm34/48/84) fused cell-permeable peptide TATm at the N-terminus, and a dominant negative mutant TmSm34/84 with stronger pro-apoptotic activity was selected and evaluated systematically in vitro. The double-site mutant of survivin (TmSm34/84) showed more robust pro-apoptotic activity against A549 cells than others, and could reverse the resistance of A549 CSCs to adriamycin (ADM) (reversal index up to 7.01) by decreasing the expression levels of survivin, P-gp, and Bcl-2 while increasing cleaved caspase-3 in CSCs. This study indicated the selected survivin dominant negative mutant TmSm34/84 is promising to be an excellent candidate for recombinant anti-cancer protein by promoting apoptosis of cancer cells and their stem cells and sensitizing chemotherapeutic drugs.

Vcp-Regulated Homologous Recombination Represents a New Druggable Vulnerability in Acute Myeloid Leukemia

Mammalian cells have developed sophisticated defense mechanisms to counteract a wide variety of stresses to which they are continuously exposed. These adaptive mechanisms are rewired in cancers, such as acute myeloid leukemia (AML), to permit oncogenic transformation (Luo J et al, Cell, 2009). Using an MLL-AF9 syngeneic mouse model, we performed a pooled in vivo shRNA screen intended to identify novel stress response vulnerabilities in AML. p97 / VCP, an AAA-ATPase protein chaperone known to be involved in protein homeostasis and ER stress, was identified as a top candidate. We first validated AML cell dependency on VCP in vivo in the MLL-AF9 model and in vitro in a panel of human AML cell lines (n=16) and primary patient samples (n=5), using VCP-directed shRNA, overexpression of a VCP dominant negative mutant or a highly selective small-molecule inhibitor of VCP, NMS-873 (Magnaghi P et al., Nat Chem Biol, 2013). The on target effect of NMS-873 in an AML context was validated using a VCP mutant (A530T), which confers resistance to VCP inhibition. We next sought to dissect the molecular mechanism by which VCP is essential to AML cell survival and proliferation. Unexpectedly, we determined that targeting VCP did not impair AML cell viability through alteration of the "proteotoxic stress" response (no accumulation of polyubiquitinilated proteins, no consistent change in proteasomal enzymatic activities and no correlation of NMS-873 sensitivity to bortezomib sensitivity in a panel of 16 AML cell lines). Using a VCP dominant negative mutant unable to translocate into the nucleus, we demonstrated that the inhibition of the nuclear, but not the cytoplasmic, fraction of VCP was sufficient to abrogate leukemic cell viability. To define new potential interacting partners of VCP that could explain its pro-leukemogenic function, we used an immunoprecipitation-mass spectrometry approach in the MV4-11 AML cell line and established by pathway overlapping analysis a significant enrichment of DNA repair pathways among the VCP protein interactome network in AML cells. Further analysis confirmed DNA damage induction through gH2AX accumulation in response to VCP inhibition and a marked reduction of homologous recombination (HR) measured using flow cytometry-based reporter assays. In further support of VCP's role in HR signaling, VCP inhibition blocked activation of the serine/threonine kinase ATM and its direct downstream targets (BRCA1, SMC1, and KAP1) in response to DNA damage induction by etoposide in AML. Indeed, the pattern of sensitivity of a panel of 16 AML cell lines and 16 primary patient samples to an ATM inhibitor, KU-55933, was highly correlated with sensitivity to the VCP inhibitor (Spearman score 0.78 and 0.72, respectively). In conclusion, we identified and validated VCP as a druggable dependency in AML and dissected the mechanistic underpinnings of VCP's role in HR orchestration through activation of ATM. Disclosures DeAngelo: Amgen: Consultancy, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Shire: Honoraria; ARIAD: Consultancy, Research Funding; Blueprint Medicines: Honoraria, Research Funding; Celgene: Research Funding; BMS: Consultancy; Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Incyte: Consultancy, Honoraria; Glycomimetics: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Immunogen: Honoraria, Research Funding. Stone: Janssen: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ono: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Seattle genetics: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Membership on an entity's Board of Directors or advisory committees; Argenix: Other: DSMB; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees; Orsenix: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Cornerstone: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Sumitomo: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Arog: Membership on an entity's Board of Directors or advisory committees, Research Funding; Actinium: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Hermine: Hybrigenics: Research Funding; Novartis: Research Funding; Celgene: Research Funding; INatherys: Equity Ownership, Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding. Stegmaier: Novartis: Consultancy, Research Funding.

Isolation and molecular characterization of a urease-negative Actinobacillus pleuropneumoniae mutant

An atypical urease-negative mutant of Actinobacillus pleuropneumoniae serovar 2 was isolated in Japan. Nucleotide sequence analysis of the urease gene cluster revealed that the insertion of a short DNA sequence into the cbiM gene was responsible for the urease-negative activity of the mutant. Veterinary diagnostic laboratories should be watchful for the presence of aberrant urease-negative A. pleuropneumoniae isolates.