V-ATPase V0a1 promotes Weibel-Palade body biogenesis through the regulation of membrane fission.

Membrane fission, the division of a membrane-bound structure into two discrete compartments, is essential for diverse cellular events, such as endocytosis and vesicle/granule biogenesis; however, the process remains unclear. The hemostatic protein von Willebrand factor is produced in vascular endothelial cells and packaged into specialized secretory granules, Weibel-Palade bodies (WPBs) at the trans-Golgi network (TGN). Here, we reported that V0a1, a V-ATPase component, is required for the membrane fission of WPBs. We identified two V0a isoforms in distinct populations of WPBs in cultured endothelial cells, V0a1 and V0a2, on mature and nascent WPBs, respectively. Although WPB buds were formed, WPBs could not separate from the TGN in the absence of V0a1. Screening using dominant-negative forms of known membrane fission regulators revealed protein kinase D (PKD) as an essential factor in biogenesis of WPBs. Further, we showed that the induction of wild-type PKDs in V0a1-depleted cells does not support the segregation of WPBs from the TGN; suggesting a primary role of V0a1 in the membrane fission of WPBs. The identification of V0a1 as a new membrane fission regulator should facilitate the understanding of molecular events that enable membrane fission.

TS-1 add-on therapy in Japanese patients with triple-negative breast cancer after neoadjuvant or adjuvant chemotherapy: a feasibility study.

Purpose We examined the feasibility, efficacy, and safety of TS-1 add-on therapy (TAT) in Japanese patients with triple-negative breast caner (TNBC). Methods TAT (TS-1, 80 mg/m2/day, BID, PO), consisting of the 21-day cycles of 14-day consecutive administration followed by 7-day drug holiday, was conducted for 365 days. The median follow-up was 75.2 months (range, 7.3-103.3 months). The primary endpoint was the feasibility of TAT. The secondary endpoints included relapse-free survival (RFS), overall survival (OS), and safety. Results 63 Japanese patients with TNBC (median age, 52.5 years; range, 23.7-68.6 years) were examined. Among them, 34 (54.0%) were postmenopausal, 54 (93.7%) had TNBC of common histological type, 51 (81.0%) had T1 to 3 tumors, 63 (100%) had undergone standardized surgery, and 44 (69.8%) and 19 (30.2%) had undergone neoadjuvant chemotherapy and adjuvant chemotherapy, respectively. The 365-day cumulative rate of TS-1 administration was 68.3% (95% confidence interval, 55.3-79.4), being comparable to 65.8% previously reported for gastric cancer. The 5-year RFS rates were 52.3% and 84.2% in the neoadjuvant and adjuvant chemotherapy groups, respectively, and the 5-year OS rates were 68.0% and 89.5%, respectively. The most common adverse events (AEs) were leucocyte count decreased (50.8%), total bilirubin decreased (44.4%), and pigmentation (42.9%). AEs were manageable clinically, and any grade 4 AEs did not develop. Conclusions The 365-day cumulative rate of TS-1 administration in TNBC patients was comparable to that in gastric cancer patients despite previous chemotherapy with anthracyclines and/or taxanes. TAT was feasible for TNBC patients after standard primary therapy.

Novel Mechanisms of ALK Activation Revealed by Analysis of the Y1278S Neuroblastoma Mutation.

Numerous mutations have been observed in the Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) in both germline and sporadic neuroblastoma. Here, we have investigated the Y1278S mutation, observed in four patient cases, and its potential importance in the activation of the full length ALK receptor. Y1278S is located in the 1278-YRASYY-1283 motif of the ALK activation loop, which has previously been reported to be important in the activation of the ALK kinase domain. In this study, we have characterized activation loop mutations within the context of the full length ALK employing cell culture and Drosophila melanogaster model systems. Our results show that the Y1278S mutant observed in patients with neuroblastoma harbors gain-of-function activity. Secondly, we show that the suggested interaction between Y1278 and other amino acids might be of less importance in the activation process of the ALK kinase than previously proposed. Thirdly, of the three individual tyrosines in the 1278-YRASYY-1283 activation loop, we find that Y1283 is the critical tyrosine in the activation process. Taken together, our observations employing different model systems reveal new mechanistic insights on how the full length ALK receptor is activated and highlight differences with earlier described activation mechanisms observed in the NPM-ALK fusion protein, supporting a mechanism of activation more in line with those observed for the Insulin Receptor (InR).

Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs.

The apical and basolateral membranes of epithelia are insulated from each other, preventing the transfer of extracellular proteins from one side to the other. Thus, a signalling protein produced apically is not expected to reach basolateral receptors. Evidence suggests that Wingless, the main Drosophila Wnt, is secreted apically in the embryonic epidermis. However, in the wing imaginal disc epithelium, Wingless is mostly seen on the basolateral membrane where it spreads from secreting to receiving cells. Here we examine the apico-basal movement of Wingless in Wingless-producing cells of wing imaginal discs. We find that it is presented first on the apical surface before making its way to the basolateral surface, where it is released and allowed to interact with signalling receptors. We show that Wingless transcytosis involves dynamin-dependent endocytosis from the apical surface. Subsequent trafficking from early apical endosomes to the basolateral surface requires Godzilla, a member of the RNF family of membrane-anchored E3 ubiquitin ligases. Without such transport, Wingless signalling is strongly reduced in this tissue.

FAM150A and FAM150B are activating ligands for anaplastic lymphoma kinase.

Aberrant activation of anaplastic lymphoma kinase (ALK) has been described in a range of human cancers, including non-small cell lung cancer and neuroblastoma (Hallberg and Palmer, 2013). Vertebrate ALK has been considered to be an orphan receptor and the identity of the ALK ligand(s) is a critical issue. Here we show that FAM150A and FAM150B are potent ligands for human ALK that bind to the extracellular domain of ALK and in addition to activation of wild-type ALK are able to drive 'superactivation' of activated ALK mutants from neuroblastoma. In conclusion, our data show that ALK is robustly activated by the FAM150A/B ligands and provide an opportunity to develop ALK-targeted therapies in situations where ALK is overexpressed/activated or mutated in the context of the full length receptor.

Tumour-associated mutations of PA-TM-RING ubiquitin ligases RNF167/RNF13 identify the PA domain as a determinant for endosomal localization.

Diverse cellular processes depend on endocytosis, intracellular vesicle trafficking, sorting and exocytosis, and processes that are regulated post-transcriptionally by modifications such as phosphorylation and ubiquitylation. The PA (protease-associated) domain E3 ligases, such as GodzillaCG10277 in Drosophila melanogaster and RNF167 (RING finger protein 167) in humans, have been implicated in the regulation of cellular endosome trafficking. In the present study, we have characterized point mutations in the RING (really interesting new gene) domain of human RNF13 and RNF167, which have been identified in human tumour samples, that abrogate ubiquitin ligase activity as well as function. In the present study, we have also identified a functional role for the PA domain, which is required for endosomal localization of these proteins. Although the PA domain point mutations of RNF13 and RNF167 identified in human tumours are ligase active, the resultant mutant proteins are mislocalized within the cell. Thus the PA domain E3 ligases examined in the present study appear to require both E3 ligase activity as well as an intact PA domain to efficiently target and ubiquitylate their cellular substrates.

Jeb/Alk signalling regulates the Lame duck GLI family transcription factor in the Drosophila visceral mesoderm.

The Jelly belly (Jeb)/Anaplastic Lymphoma Kinase (Alk) signalling pathway regulates myoblast fusion in the circular visceral mesoderm (VM) of Drosophila embryos via specification of founder cells. However, only a limited number of target molecules for this pathway are described. We have investigated the role of the Lame Duck (Lmd) transcription factor in VM development in relationship to Jeb/Alk signal transduction. We show that Alk signalling negatively regulates Lmd activity post-transcriptionally through the MEK/MAPK (ERK) cascade resulting in a relocalisation of Lmd protein from the nucleus to cytoplasm. It has previously been shown that downregulation of Lmd protein is necessary for the correct specification of founder cells. In the visceral mesoderm of lmd mutant embryos, fusion-competent myoblasts seem to be converted to 'founder-like' cells that are still able to build a gut musculature even in the absence of fusion. The ability of Alk signalling to downregulate Lmd protein requires the N-terminal 140 amino acids, as a Lmd(141-866) mutant remains nuclear in the presence of active ALK and is able to drive robust expression of the Lmd downstream target Vrp1 in the developing VM. Our results suggest that Lmd is a target of Jeb/Alk signalling in the VM of Drosophila embryos.

Goliath family E3 ligases regulate the recycling endosome pathway via VAMP3 ubiquitylation.

Diverse cellular processes depend on endocytosis, intracellular vesicle trafficking, sorting and exocytosis, processes regulated post-transcriptionally by modifications such as phosphorylation and ubiquitylation. In addition to sorting to the lysosome, cargo is recycled to the plasma membrane via recycling endosomes. Here, we describe a role of the goliath gene family of protease-associated (PA) domain E3 ligases in regulating recycling endosome trafficking. The two Drosophila members of this family--Goliath and Godzilla(CG10277)--are located on endosomes, and both ectopic expression and loss-of-function lead to the accumulation of Rab5-positive giant endosomes. Furthermore, the human homologue RNF167 exhibits similar behaviour. We show that the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) protein VAMP3 is a target of these ubiquitin ligases, and that recycling endosome trafficking is abrogated in response to their activity. Furthermore, mutation of the Godzilla ubiquitylation target lysines on VAMP3 abrogates the formation of enlarged endosomes induced by either Godzilla or RNF167. Thus, Goliath ubiquitin ligases play a novel role in regulating recycling endosome trafficking via ubiquitylation of the VAMP3 SNARE protein.

Cell culture and Drosophila model systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma.

Neuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALK(F1174) mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALK(F1174l), (ii) kinase-dead ALK mutants, e.g. ALK(I1250T) (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.

Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684.

Mutations in the kinase domain of ALK (anaplastic lymphoma kinase) have recently been shown to be important for the progression of the childhood tumour neuroblastoma. In the present study we investigate six of the putative reported constitutively active ALK mutations, in positions G1128A, I1171N, F1174L, R1192P, F1245C and R1275Q. Our analyses were performed in cell-culture-based systems with both mouse and human ALK mutant variants and subsequently in a Drosophila melanogaster model system. Our investigation addressed the transforming potential of the putative gain-of-function ALK mutations as well as their signalling potential and the ability of two ATP-competitive inhibitors, Crizotinib (PF-02341066) and NVP-TAE684, to abrogate the activity of ALK. The results of the present study indicate that all mutations tested are of an activating nature and thus are implicated in tumour initiation or progression of neuroblastoma. Importantly for neuroblastoma patients, all ALK mutations used in the present study can be blocked by the inhibitors, although some mutants exhibited higher levels of drug sensitivity than others.

The neuroblastoma ALK(I1250T) mutation is a kinase-dead RTK in vitro and in vivo.

Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) have recently been shown to be an important determinant in the genetics of the childhood tumor neuroblastoma. Here we discuss an in-depth analysis of one of the reported gain-of-function ALK mutations-ALK(I1250T)-identified in the germ line DNA of one patient. Our analyses were performed in cell culture-based systems and subsequently confirmed in a Drosophila model. The results presented here indicate that the germ line ALK(I1250T) mutation is most probably not a determinant for tumor initiation or progression and, in contrast, seems to generate a kinase-dead mutation in the ALK receptor tyrosine kinase (RTK). Consistent with this, stimulation with agonist ALK antibodies fails to lead to stimulation of ALK(I1250T) and we were unable to detect tyrosine phosphorylation under any circumstances. In agreement, ALK(I1250T) is unable to activate downstream signaling pathways or to mediate neurite outgrowth, in contrast to the activated wild-type ALK receptor or the activating ALK(F1174S) mutant. Identical results were obtained when the ALK(I1250T) mutant was expressed in a Drosophila model, confirming the lack of activity of this mutant ALK RTK. We suggest that the ALK(I1250T) mutation leads to a kinase-dead ALK RTK, in stark contrast to assumed gain-of-function status, with significant implications for patients reported to carry this particular ALK mutation.

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption.

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 µM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 µM amiloride and that recombinant αßγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 µM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the ß(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

Re-evaluation of M-LAO, L-amino acid oxidase, from the venom of Gloydius blomhoffi as an anticoagulant protein.

Many anticoagulant proteins have been found from snake venoms. Recently, (L)-amino acid oxidase (LAO) from the venom of Gloydius blomhoffi, M-LAO, was reported to inhibit coagulation factor IX; however, the mechanism of its anticoagulant activity is still unclear. Here, we re-evaluated the anticoagulant activity of M-LAO. We first purified M-LAO from the venom of G. blomhoffi, and examined the effect of LAO inhibitors and the hydrogen peroxide scavenger, catalase, on the anticoagulant activity of M-LAO. We found that the isolated M-LAO fraction prolongs the APTT, PT and fibrinogen clotting time and cleaves the Aalpha-chain of fibrinogen. LAO inhibitors or catalase did not inhibit these effects. Detailed analysis revealed that the M-LAO fraction contained a small amount of 39-kDa metalloproteinase. The prolongation of clotting time and degradation of fibrinogen were inhibited by a metalloproteinase inhibitor. Therefore, we concluded that the anticoagulant activity of the M-LAO fraction was caused by the 39-kDa metalloproteinase.

Snake venom Vascular Endothelial Growth Factors (VEGF-Fs) exclusively vary their structures and functions among species.

Vascular endothelial growth factor (VEGF-A) and its family proteins are crucial regulators of blood vessel formation and vascular permeability. Snake venom has recently been shown to be an exogenous source of unique VEGF (known as VEGF-F), and now, two types of VEGF-F with distinct biochemical properties have been reported. Here, we show that VEGF-Fs (venom-type VEGFs) are highly variable in structure and function among species, in contrast to endogenous tissue-type VEGFs (VEGF-As) of snakes. Although the structures of tissue-type VEGFs are highly conserved among venomous snake species and even among all vertebrates, including humans, those of venom-type VEGFs are extensively variegated, especially in the regions around receptor-binding loops and C-terminal putative coreceptor-binding regions, indicating that highly frequent variations are located around functionally key regions of the proteins. Genetic analyses suggest that venom-type VEGF gene may have developed from a tissue-type gene and that the unique sequence of its C-terminal region was generated by an alteration in the translation frame in the corresponding exons. We further verified that a novel venom-type VEGF from Bitis arietans displays unique properties distinct from already known VEGFs. Our results may provide evidence of a novel mechanism causing the generation of multiple snake toxins and also of a new model of molecular evolution.

VEGF-A and VEGF-F evoke distinct changes in vascular ultrastructure.

VEGF-induced vascular barrier disruptions, formation of fenestrae and vesiculo-vacuolar organelles (VVOs), are involved in enhancing vascular permeability in metastatic and edematous diseases. Here, we analyzed vascular permeability enhanced by VEGFs with different receptor selectivity using biological and ultrastructural methods. VEGF-A(165), which stimulates both Flt-1 and KDR, induced the formation of both fenestrae and VVOs at a similar rate, while VEGF-F, a KDR-specific VEGF vammin, predominantly induced the formation of fenestrae with an approximately 5-fold more potent vascular permeability response than VEGF-A(165). This is the first report showing that VEGFs with different receptor selectivity evoke distinct changes in vascular ultrastructure.

Anticoagulant mechanism of factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis.

Anticoagulant mechanism of the coagulation factor IX/factor X-binding protein (IX/X-bp) isolated from the venom of Trimeresurus flavoviridis was investigated. IX/X-bp had no effect on the amidase activity of factor Xa measured with a synthetic peptide substrate Boc-Leu-Gly-Arg-pNA. Prothrombin activation by factor Xa without cofactors, such as factor Va and phospholipids, was only slightly influenced by IX/X-bp. However, prothrombin activation by factor Xa in the presence of factor Va resulted in IX/X-bp inhibiting the increase of k(cat) of thrombin formation through inhibition of interaction between factor Xa and factor Va. IX/X-bp also inhibited the decrease of K(m) for thrombin formation through interaction with phospholipids. Thus, IX/X-bp appears to act as an anticoagulant protein by inhibiting the interaction between factor Xa and its cofactors in the prothrombinase complex by binding to the Gla domain of factor Xa.

Structural divergence of cysteine-rich secretory proteins in snake venoms.

Cysteine-rich secretory proteins (CRISPs) are expressed in spermatocytes and granules of neutrophils in mammals, and are associated with sperm maturation and host defense. Related proteins have recently been recovered in snake venoms, and some of the snake venom-derived CRISPs exhibit ion channel blocking activity. Here we isolated and identified two novel CRISPs (kaouthin-1 and kaouthin-2) from the venom of Naja kaouthia (Elapidae), and cloned the encoding cDNAs. Kaouthin-1 and kaouthin-2 were classified into two broad sister groups of Elapidae, the Asian species and the marine/Australian species, respectively. Sequence comparisons reveal that the high-frequency variable regions among snake venom CRISPs define a continuous line on the molecular surface of the N-terminal pathogenesis-related protein-1 (PR-1) domain and the C-terminal cysteine-rich domain (CRD). Snake venom proteins generally display efficient molecular diversity around functionally key regions, suggesting that the PR-1 domain of CRISPs is important for the recognition of target molecules.