Analysis of the mechanisms controlling the activity of flp1, a chromosomal passenger protein in the fission Schizosaccharomyces pombe

ABSTRACT In S. cerevisiae, the protein phosphatase Cdc14pwt is essential far mitotic exit through its contribution to reducing mitotic CDK activity. But Cdc14pwt also acts as a mare general temporal coordinator of mid and late mitotic events by controlling the partitioning of DNA, microtubule stability and cytokinesis. Cdc14pwt orthologs are well conserved from yeasts to humans, and sequence comparison revealed the presence of three domains, A, B and C, of which A and B form the catalytic domain. Cdc14pwt orthologs are regulated (in part) through cell cycle dependent changes in their localization. Some of them are thought to be kept inactive by sequestration in the nucleolus during interphase. This is the case for flp1pwt, the single identified Cdc14pwt ortholog in the fission yeast S. pombe. In early mitosis, flp1pwt leaves the nucleolus and localizes to the kinetochores, the contractile ring and the mitotic spindle, suggesting that it has multiple substrates and regulates many mitotic processes. flp1D cells show a high chromosome loss rate and septation defects, suggesting a role for flp1wt in the fidelity of chromosome transmission and cytokinesis. The aim of this study is to characterize the mechanisms underlying flp1pwt functions and the control of its activity. A structure-function analysis has revealed that the presence of both A and B domains is required for biological function and for proper flp1pwt mitotic localization. In contrast, the C domain of flp1pwt is responsible for its proper nucleolar localization in G2/interphase. My data suggest that dephosphorylation of substrates by flp1pwt is not necessary for any changes in localization of flp1pwt except that at the medial ring. In that particular case, the catalytic activity of flp1pwt is required for efficient localization, therefore revealing an additional level of regulation. All the functions of flp1pwt assayed to date require its catalytic activity, emphasizing the importance of further identification of its substrates. As described for other orthologs, the capability of selfinteraction and phosphorylation status might help to control flp1pwt activity. My data suggest that flp1pwt forms oligomers in vivo and that phosphorylation is not essential far localization changes of the protein. In addition, the hypophosphorylated form of flp1pwt might be specifically involved in the promotion of cytokinesis. The results of this study suggest that multiple modes of regulation including localization, selfassociation and phosphorylation allow a fine-tuning regulation of flp1pwt phosphatase activity, and more generally that of Cdc14pwt family of phosphatases. RESUME Chez la levure S. cerevisiae, la protéine phosphatase Cdc14pwt est essentielle pour la sortie de mitose du fait de sa contribution dans la réduction d'activité des CDK mitotiques. Comme elle contrôle également le partage de l'ADN, la stabilité des microtubules et la cytokinèse, Cdc14pwt est en fait considérée comme un coordinateur temporel général des évènements de milieu et de fin de mitose. Les orthologues de Cdc14pwt sont bien conservés, des levures jusqu'à l'espèce humaine. Des comparaisons de séquence ont révélé la présence de trois domaines A, B et C, les deux premiers constituant le domaine catalytique. Ils sont régulés (en partie) via des changements dans leur localisation, eux-mêmes dépendants du cycle cellulaire. Plusieurs de ces orthologues sont supposés inactivés par séquestration dans le nucléole en interphase, ce qui est le cas de flp1pwt le seul orthologue de Cdc14pwt identifié chez la levure fissipare S, pombe. En début de mitose, flp1pwt quitte le nucléole et localise au niveau des kinetochores, de l'anneau contractile d'actine et du fuseau mitotique, ce qui laisse supposer de multiples substrats et fonctions. Comme les cellules délétées pour le gène flp1wt présentent un taux élevé de perte de chromosome et des défauts de septation, flp1pwt semble jouer un rôle dans la fidélité de la transmission du matériel génétique et la cytokinèse. Le but de cette étude est de caractériser les mécanismes impliqués dans les fonctions assurées par flp1pwt d'une part, et dans le contrôle de son activité d'autre part. Une analyse structure-fonction a révélé que la présence simultanée des deux domaines A et B est requise pour la fonction biologique de flp1pwt et sa localisation correcte pendant la mitose. Par contre, le domaine C de flp1pwt confère une localisation nucléolaire adéquate en G2/interphase. Mes données suggèrent que la déphosphorylation de substrats par flp1pwt est dispensable pour sa localisation correcte excepté celle à l'anneau médian, qui requiert dans ce cas, l'activité catalytique de flp1pwt, révélant ainsi un niveau de régulation supplémentaire. Toutes les fonctions de flp1 pwt testées jusqu'à présent nécessitent également son activité catalytique, ce qui accentue l'importance de l'identification future de ses substrats. Comme cela a déjà été décrit pour d'autres orthologues, la capacité d'auto-intéraction et le niveau de phosphorylation pourraient contrôler l'activité de flp1pwt. En effet, mes données suggèrent que flp1pwt forme des oligomères in vivo et que la phosphorylation n'est pas essentielle pour les changements de localisation observés pour la protéine. De plus, la forme hypophosphorylée de flp1pwt pourrait être spécifiquement impliquée dans la promotion de la cytokinèse. De multiples modes de régulation incluant la localisation, l'auto-association et la phosphorylation semblent permettre un contrôle fin et subtil de l'activité de la phosphatase flp1pwt, et plus généralement celle des protéines de la famille de Cdc14pwt.

Cytosolic sensing of extracellular self-DNA transported into monocytes by the antimicrobial peptide LL37.

The intracellular location of nucleic acid sensors prevents recognition of extracellular self-DNA released by dying cells. However, on forming a complex with the endogenous antimicrobial peptide LL37, extracellular DNA is transported into endosomal compartments of plasmacytoid dendritic cells, leading to activation of Toll-like receptor-9 and induction of type I IFNs. Whether LL37 also transports self-DNA into nonplasmacytoid dendritic cells, leading to type I IFN production via other intracellular DNA receptors is unknown. Here we found that LL37 very efficiently transports self-DNA into monocytes, leading the production of type I IFNs in a Toll-like receptor-independent manner. This type I IFN induction was mediated by double-stranded B form DNA, regardless of its sequence, CpG content, or methylation status, and required signaling through the adaptor protein STING and TBK1 kinase, indicating the involvement of cytosolic DNA sensors. Thus, our study identifies a novel link between the antimicrobial peptides and type I IFN responses involving DNA-dependent activation of cytosolic sensors in monocytes.

A soluble form of B cell maturation antigen, a receptor for the tumor necrosis factor family member APRIL, inhibits tumor cell growth.

A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.

Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes.

The human estrogen receptor (hER) is a trans-acting regulatory protein composed of a series of discrete functional domains. We have microinjected an hER expression vector (HEO) into Xenopus oocyte nuclei and demonstrate, using Western blot assay, that the hER is synthesized. When nuclear extracts from oocytes were prepared and incubated in the presence of a 2.7 kb DNA fragment comprising the 5' end of the vitellogenin gene B2, formation of estrogen-dependent complexes could be visualized by electron microscopy over the estrogen responsive element (ERE). Of crucial importance is the observation that the complex formation is inhibited by the estrogen antagonist tamoxifen, is restored by the addition of the hormone and does not take place with extracts from control oocytes injected with the expression vector lacking the sequences encoding the receptor. The presence of the biologically active hER is confirmed in co-injection experiments, in which HEO is co-introduced with a CAT reporter gene under the control of a vitellogenin promoter containing or lacking the ERE. CAT assays and primer extensions analyses reveal that both the receptor and the ERE are essential for estrogen induced stimulation of transcription. The same approach was used to analyze selective hER mutants. We find that the DNA binding domain (region C) is essential for protein--DNA complex formation at the ERE but is not sufficient by itself to activate transcription from the reporter gene. In addition to region C, both the hormone binding (region E) and amino terminal (region A/B) domains are needed for an efficient transcription activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Dukes B colorectal cancer: distinct genetic categories and clinical outcome based on proximal or distal tumor location.

PURPOSE: The aim of this study was to determine whether tumor location proximal or distal to the splenic flexure is associated with distinct molecular patterns and can predict clinical outcome in a homogeneous group of patients with Dukes B (T3-T4, N0, M0) colorectal cancer. It has been hypothesized that proximal and distal colorectal cancer may arise through different pathogenetic mechanisms. Although p53 and Ki-ras gene mutations occur frequently in distal tumors, another form of genomic instability associated with defective DNA mismatch repair has been predominantly identified in the proximal colon. To date, however, the clinical usefulness of these molecular characteristics remains unproven. METHODS: A total of 126 patients with a lymph node-negative sporadic colon or rectum adenocarcinoma were prospectively assessed with the endpoint of death by cancer. No patient received either radiotherapy or chemotherapy. p53 protein was studied by immunohistochemistry using DO-7 monoclonal antibody, and p53 and Ki-ras gene mutations were detected by single strand conformation polymorphism assay. RESULTS: During a mean follow-up of 67 months, the overall five-year survival was 70 percent. Nuclear p53 staining was found in 57 tumors (47 percent), and was more frequent in distal than in proximal tumors (55 vs. 21 percent; chi-squared test, P < 0.001). For the whole group, p53 protein expression correlated with poor survival in univariate and multivariate analysis (log-rank test, P = 0.01; hazard ratio = 2.16; 95 percent confidence interval = 1.12-4.11, P = 0.02). Distal colon tumors and rectal tumors exhibited similar molecular patterns and showed no difference in clinical outcome. In comparison with distal colorectal cancer, proximal tumors were found to be statistically significantly different on the following factors: mucinous content (P = 0.008), degree of histologic differentiation (P = 0.012), p53 protein expression, and gene mutation (P = 0.001 and 0.01 respectively). Finally, patients with proximal tumors had a marginally better survival than those with distal colon or rectal cancers (log-rank test, P = 0.045). CONCLUSION: In this series of Dukes B colorectal cancers, p53 protein expression was an independent factor for survival, which also correlated with tumor location. Eighty-six percent of p53-positive tumors were located in the distal colon and rectum. Distal colon and rectum tumors had similar molecular and clinical characteristics. In contrast, proximal neoplasms seem to represent a distinct entity, with specific histopathologic characteristics, molecular patterns, and clinical outcome. Location of the neoplasm in reference to the splenic flexure should be considered before group stratification in future trials of adjuvant chemotherapy in patients with Dukes B tumors.

Transcriptional activation of the utrophin promoter B by a constitutively active Ets-transcription factor.

Duchenne muscular dystrophy is an X-linked genetic disease caused by the absence of functional dystrophin. Pharmacological upregulation of utrophin, the autosomal homologue of dystrophin, offers a potential therapeutic approach to treat Duchenne patients. Full-length utrophin mRNA is transcribed from two alternative promoters, called A and B. In contrast to the utrophin promoter A, little is known about the factors regulating the activity of the utrophin promoter B. Computer analysis of this second promoter revealed the presence of several conserved binding motives for Ets-transcription factors. Using electrotransfer of cDNA into mouse muscles, we demonstrate that a genetically modified beta-subunit of the Ets-transcription factor GA-binding protein potently activates a utrophin promoter B reporter construct in innervated muscle fibers in vivo. These results make the GA-binding protein and the signaling cascade regulating its activity in muscle cells, potential targets for the pharmacological modulation of utrophin expression in Duchenne patients.

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.

A-kinase-anchoring protein-Lbc anchors IκB kinase β to support interleukin-6-mediated cardiomyocyte hypertrophy.

In response to stress, the heart undergoes a pathological remodeling process associated with hypertrophy and the reexpression of a fetal gene program that ultimately causes cardiac dysfunction and heart failure. In this study, we show that A-kinase-anchoring protein (AKAP)-Lbc and the inhibitor of NF-κB kinase subunit β (IKKβ) form a transduction complex in cardiomyocytes that controls the production of proinflammatory cytokines mediating cardiomyocyte hypertrophy. In particular, we can show that activation of IKKβ within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukin-6 (IL-6), which in turn enhances fetal gene expression and cardiomyocyte growth. These findings provide a new mechanistic hypothesis explaining how hypertrophic signals are coordinated and conveyed to interleukin-mediated transcriptional reprogramming events in cardiomyocytes.

A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability.

FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

Activities of fluconazole, caspofungin, anidulafungin, and amphotericin B on planktonic and biofilm Candida species determined by microcalorimetry.

We investigated the activities of fluconazole, caspofungin, anidulafungin, and amphotericin B against Candida species in planktonic form and biofilms using a highly sensitive assay measuring growth-related heat production (microcalorimetry). C. albicans, C. glabrata, C. krusei, and C. parapsilosis were tested, and MICs were determined by the broth microdilution method. The antifungal activities were determined by isothermal microcalorimetry at 37°C in RPMI 1640. For planktonic Candida, heat flow was measured in the presence of antifungal dilutions for 24 h. Candida biofilm was formed on porous glass beads for 24 h and exposed to serial dilutions of antifungals for 24 h, and heat flow was measured for 48 h. The minimum heat inhibitory concentration (MHIC) was defined as the lowest antifungal concentration reducing the heat flow peak by ≥50% (≥90% for amphotericin B) at 24 h for planktonic Candida and at 48 h for Candida biofilms (measured also at 24 h). Fluconazole (planktonic MHICs, 0.25 to >512 μg/ml) and amphotericin B (planktonic MHICs, 0.25 to 1 μg/ml) showed higher MHICs than anidulafungin (planktonic MHICs, 0.015 to 0.5 μg/ml) and caspofungin (planktonic MHICs, 0.125 to 0.5 μg/ml). Against Candida species in biofilms, fluconazole's activity was reduced by >1,000-fold compared to its activity against the planktonic counterparts, whereas echinocandins and amphotericin B mainly preserved their activities. Fluconazole induced growth of planktonic C. krusei at sub-MICs. At high concentrations of caspofungin (>4 μg/ml), paradoxical growth of planktonic C. albicans and C. glabrata was observed. Microcalorimetry enabled real-time evaluation of antifungal activities against planktonic and biofilm Candida organisms. It can be used in the future to evaluate new antifungals and antifungal combinations and to study resistant strains.

Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia.

A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed "autosis," has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia-ischemia in vivo. A chemical screen of ~5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na(+),K(+)-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na(+),K(+)-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na(+),K(+)-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.

PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block

Acquired form of angioedema of the head and neck related to a deficiency in c1-inhibitor: a case report with a review of the literature.

Angioedema related to a deficiency in the C1-inhibitor protein is characterized by its lack of response to therapies including antihistamine, steroids, and epinephrine. In the case of laryngeal edema, mortality rate is approximately 30 percent. The first case of the acquired form of angioedema related to a deficiency in C1-inhibitor was published in 1972. In our paper, we present a case of an acquired form of angioedema of the oropharyngeal region secondary to the simultaneous occurrence of two causative factors: neutralization of C1-inhibitor by an autoantibody and the use of an angiotensin convertin enzyme inhibitor.

Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling.

Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.