VPS4B deficiency causes early embryonic lethality and induces signal transduction disorders of cell endocytosis.

VPS4B (vacuolar protein sorting 4B), a member of the ATPase associated with diverse cellular activities (AAA) protein family, is a component of the endosomal sorting complexes required for transport machinery which regulates the internalization and lysosomal degradation of membrane proteins. We previously reported that VPS4B is one of the pathogenic genes related to dentin dysplasia type I, although its function was largely unknown. To investigate the role of VPS4B in tooth development, we deleted the Vps4b gene in mice. We found that heterozygous knockout mice (Vps4b+/- ) developed normally and were fertile. However, homozygous deletion of the Vps4b gene resulted in early embryonic lethality of Vps4b-/- mice at approximately embryonic day 9.5 (E9.5). To investigate the underlying molecular mechanisms, we examined the molecular functions of VPS4B in vivo and in vitro. Cell experiments showed that VPS4B influenced the proliferation, apoptosis, and cell cycle of transfected human neuroblastoma cells (IMR-32 cells) with over-expression or knockdown of VPS4B. Moreover, qRT-PCR detection showed that the mRNA expression levels of apoptosis-, cell cycle-, and endocytosis-related genes was significantly down or up-regulated in RNA interference-mediated knockdown of VPS4B in IMR-32 cells and Vps4b+/- E12.5 embryos. We accordingly speculated that signal transduction disorders of cell endocytosis are a contributing factor to the prenatal lethality of Vps4b-/- mice.

ESCRT-III controls nuclear envelope reformation.

During telophase, the nuclear envelope (NE) reforms around daughter nuclei to ensure proper segregation of nuclear and cytoplasmic contents. NE reformation requires the coating of chromatin by membrane derived from the endoplasmic reticulum, and a subsequent annular fusion step to ensure that the formed envelope is sealed. How annular fusion is accomplished is unknown, but it is thought to involve the p97 AAA-ATPase complex and bears a topological equivalence to the membrane fusion event that occurs during the abscission phase of cytokinesis. Here we show that the endosomal sorting complex required for transport-III (ESCRT-III) machinery localizes to sites of annular fusion in the forming NE in human cells, and is necessary for proper post-mitotic nucleo-cytoplasmic compartmentalization. The ESCRT-III component charged multivesicular body protein 2A (CHMP2A) is directed to the forming NE through binding to CHMP4B, and provides an activity essential for NE reformation. Localization also requires the p97 complex member ubiquitin fusion and degradation 1 (UFD1). Our results describe a novel role for the ESCRT machinery in cell division and demonstrate a conservation of the machineries involved in topologically equivalent mitotic membrane remodelling events.

VPS-22/SNF8 regulates longevity via modulating the activity of DAF-16 in C. elegans.

Aging is regulated by complex signaling networks, the details of which remain poorly understood. Here, we demonstrate that VPS-22/SNF8, a component of endosomal sorting complex required for transport-II (ESCRT-II), regulates the lifespan of C. elegans. In this study we show that worms with vps-22/snf8 gene knockdown had a shorter lifespan than wild-type worms. The expression pattern of VPS-22/SNF8 in C. elegans was highly similar to that of DAF-16. Knockout of daf-16 in C. elegans shortened the worms' lifespan; however, reducing the expression of vps-22/snf8 in daf-16 null worms did not further shorten their lifespan, indicating that vps-22/snf8 and daf-16 may act in the same signaling pathway to regulate longevity. Over-expression of daf-16 rescued the short-lived phenotype of vps-22/snf8 knockdown worms. Moreover, down-regulation of vps-22/snf8 decreased the nuclear localization of DAF-16 and modulated the expression of daf-16 downstream genes that regulate longevity in C. elegans. In summary, our results indicate that vps-22/snf8 can regulate the longevity of C. elegans by partially modulating the activity of daf-16. These findings may help us to better understand the mechanisms of aging.

The ESCRT System Plays an Important Role in the Germination in Candida albicans by Regulating the Expression of Hyphal-Specific Genes and the Localization of Polarity-Related Proteins.

Candida albicans is an important opportunistic fungal pathogen, and its pathogenicity is closely related to its ability to form hyphae. ESCRT system was initially discovered as a membrane-budding machinery involved in the formation of multivesicular bodies. More recently, the role of ESCRT is vastly expanded. Early reports showed that the ESCRT system is involved in inducing hyphae under neutral-alkaline environment via the Rim101 pathway. We previously found that in the environment that contains serum, one ESCRT protein, Vps4, is essential for polarity maintenance during hyphal formation, as its deletion causes the formation of multiple hyphae. In this study, we found that Vps4 is also essential for the proper localization of Cdc42 and Cdc3, which may be related to its role in polarity maintenance. We also discovered that deletions of the ESCRT proteins significantly delay germination and cause downregulation of hyphal-specific genes, most prominent of which is HGC1. Since Hgc1 is essential for many aspects of hyphal growth, its downregulation could explain our observed phenotypes. Our further studies show that ESCRT proteins are involved in the dynamics of Ras1. Deletions of VPS4 or SNF7 significantly decrease the recovery rate of GFP-Ras1 in the fluorescence recovery after photobleaching experiment. The decreased Ras1 dynamics may disrupt the signaling pathway and lead to downregulation of hyphal-specific genes. Therefore, in this study we discovered a novel and Rim101 independent mechanism used by the ESCRT system to regulate hyphal induction and polarity maintenance, which could provide insights on the pathogenicity mechanism of Candia albicans.

Vps4b heterozygous mice do not develop tooth defects that replicate human dentin dysplasia I.

BACKGROUND: Vacuolar protein sorting-associated protein 4B (VPS4B) is a member of the ATP enzyme AAA protein family, and is mainly involved in protein degradation and cell membrane fusion. Recently, a dominant mutation in this gene was identified in human dentin dysplasia type I (DD-I). Herein, we report the generation of Vps4b knockout (Vps4b KO) mice; however, the homozygous Vps4b KO mutation was embryonic lethal at the early stages of embryo development, and we therefore report the results of heterozygous mutant mice. RESULTS: Mice heterozygous for Vps4b did not develop tooth defects replicating human DD-I. Immunohistochemistry showed that gene KO was successful, as there was decreased expression of Vps4b in heterozygous mice; hematoxylin and eosin (H&E) staining also showed that the width of the pre-dentin zone was increased in heterozygous mice, although the arrangement of the odontoblasts was not significantly different from wild-type (WT) mice. However, H&E staining showed no obvious abnormalities in the bones of heterozygous mice. Moreover, stereomicroscopic and X-ray radiography results indicated no abnormal manifestations in teeth or bones. Furthermore, statistical analysis of the volume and density of dentin and enamel, as well as skeletal analysis, including the volume and separation of trabecular bone analyzed by micro-CT, all showed no differences between Vps4b heterozygotes and WT mice. In addition, there also were no significant differences in bone or cartilage mineralization as evaluated by Alcian blue-Alizarin red staining. CONCLUSIONS: The heterozygous Vps4b KO mice do not develop tooth defects that replicate human DD-I and this is likely to be due to differences in tooth development between the two species. Consequently, further studies are needed to determine whether mice are an appropriate animal model for human tooth diseases.

Regulation of postsynaptic function by the dementia-related ESCRT-III subunit CHMP2B.

The charged multivesicular body proteins (Chmp1-7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause frontotemporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, decreased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b-containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines.

UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y regulate TDP-43 protein ubiquitination.

Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3(C145S) was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43(K263E) ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43(K263E) ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration.

The ubiquitin ligase Nedd4 regulates craniofacial development by promoting cranial neural crest cell survival and stem-cell like properties.

The integration of multiple morphogenic signalling pathways and transcription factor networks is essential to mediate neural crest (NC) cell induction, delamination, survival, stem-cell properties, fate choice and differentiation. Although the transcriptional control of NC development is well documented in mammals, the role of post-transcriptional modifications, and in particular ubiquitination, has not been explored. Here we report an essential role for the ubiquitin ligase Nedd4 in cranial NC cell development. Our analysis of Nedd4(-/-) embryos identified profound deficiency of cranial NC cells in the absence of structural defects in the neural tube. Nedd4 is expressed in migrating cranial NC cells and was found to positively regulate expression of the NC transcription factors Sox9, Sox10 and FoxD3. We found that in the absence of these factors, a subset of cranial NC cells undergo apoptosis. In accordance with a lack of cranial NC cells, Nedd4(-/-) embryos have deficiency of the trigeminal ganglia, NC derived bone and malformation of the craniofacial skeleton. Our analyses therefore uncover an essential role for Nedd4 in a subset of cranial NC cells and highlight E3 ubiquitin ligases as a likely point of convergence for multiple NC signalling pathways and transcription factor networks.

Ubiquitin-dependent regulation of phospho-AKT dynamics by the ubiquitin E3 ligase, NEDD4-1, in the insulin-like growth factor-1 response.

AKT is a critical effector kinase downstream of the PI3K pathway that regulates a plethora of cellular processes including cell growth, death, differentiation, and migration. Mechanisms underlying activated phospho-AKT (pAKT) translocation to its action sites remain unclear. Here we show that NEDD4-1 is a novel E3 ligase that specifically regulates ubiquitin-dependent trafficking of pAKT in insulin-like growth factor (IGF)-1 signaling. NEDD4-1 physically interacts with AKT and promotes HECT domain-dependent ubiquitination of exogenous and endogenous AKT. NEDD4-1 catalyzes K63-type polyubiquitin chain formation on AKT in vitro. Plasma membrane binding is the key step for AKT ubiquitination by NEDD4-1 in vivo. Ubiquitinated pAKT translocates to perinuclear regions, where it is released into the cytoplasm, imported into the nucleus, or coupled with proteasomal degradation. IGF-1 signaling specifically stimulates NEDD4-1-mediated ubiquitination of pAKT, without altering total AKT ubiquitination. A cancer-derived plasma membrane-philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4-1 and more efficiently trafficked into the nucleus compared with wild type AKT. This study reveals a novel mechanism by which a specific E3 ligase is required for ubiquitin-dependent control of pAKT dynamics in a ligand-specific manner.

Calcineurin inhibitors suppress the high-temperature stress sensitivity of the yeast ubiquitin ligase Rsp5 mutant: a new method of screening for calcineurin inhibitors.

The ubiquitin/proteasome system plays significant and important roles in the regulation of metabolism of various proteins. The dysfunction of this system is involved in several diseases, for example, cancer, neurogenic diseases and chronic inflammation. Therefore, the compounds, which regulate the ubiquitin/proteasome system, might be candidates for the development use as clinical drugs. The Saccharomyces cerevisiae mutant (rsp5(A401E)) has a single amino acid change, Ala401Glu, in the RSP5 gene, which encodes an essential E3 ubiquitin ligase, is hypersensitive to high-temperature stress. Here, we found that the immunosuppressants FK506 and cyclosporin A, both known as calcineurin inhibitors, complemented the high-temperature stress-induced growth defect of rsp5(A401E) strain. The defect of calcineurin pathway by disrupting the CNB1 and CRZ1 gene also partially complemented the high-temperature stress sensitivity of rsp5(A401E) cells. Thus, these results suggest that inhibition of the calcineurin pathway confers the tolerance to high-temperature stress on rsp5(A401E) cells. Furthermore, some diterpenoid compounds, which restore the growth of rsp5(A401E) cells, showed the activities of calcineurin inhibition and protein phosphatase 2C activation. These results indicate that calcineurin inhibitors suppress the high-temperature stress sensitivity of rsp5(A401E) cells and that analysis of their physiological function is effective for the screening of calcineurin inhibitors in yeast cells.

Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis.

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.

The Nedd4-like ubiquitin E3 ligases target angiomotin/p130 to ubiquitin-dependent degradation.

AMOT (angiomotin) is a membrane-associated protein that is expressed in ECs (endothelial cells) and controls migration, TJ (tight junction) formation, cell polarity and angiogenesis. Recent studies have revealed that AMOT and two AMOT-like proteins, AMOTL1 and AMOTL2, play critical roles in the Hippo pathway by regulating the subcellular localization of the co-activators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). However, it has been unclear how AMOT is regulated. In the present study, we report that AMOT undergoes proteasomal degradation. We identify three members of Nedd4 (neural-precursor-cell-expressed developmentally down-regulated)-like ubiquitin E3 ligases, Nedd4, Nedd4-2 and Itch, as the ubiquitin E3 ligases for the long isoform of AMOT, AMOT/p130. We demonstrate that Nedd4, Nedd4-2 and Itch mediate poly-ubiquitination of AMOT/p130 in vivo. Overexpression of Nedd4, Nedd4-2 or Itch leads to AMOT/p130 proteasomal degradation. Knockdown of Nedd4, Nedd4-2 and Itch causes an accumulation of steady-state level of AMOT/p130. We also show that three L/P-PXY motifs of AMOT/p130 and the WW domains of Nedd4 mediate their interaction. Furthermore, Nedd4-like ubiquitin E3 ligases might compete with YAP for the binding to AMOT/p130, and subsequently targeting AMOT/p130 for ubiquitin-dependent degradation. Together, these observations reveal a novel post-translational regulatory mechanism of AMOT/p130.

Human ESCRT-III and VPS4 proteins are required for centrosome and spindle maintenance.

The ESCRT pathway helps mediate the final abscission step of cytokinesis in mammals and archaea. In mammals, two early acting proteins of the ESCRT pathway, ALIX and TSG101, are recruited to the midbody through direct interactions with the phosphoprotein CEP55. CEP55 resides at the centrosome through most of the cell cycle but then migrates to the midbody at the start of cytokinesis, suggesting that the ESCRT pathway may also have centrosomal links. Here, we have systematically analyzed the requirements for late-acting mammalian ESCRT-III and VPS4 proteins at different stages of mitosis and cell division. We found that depletion of VPS4A, VPS4B, or any of the 11 different human ESCRT-III (CHMP) proteins inhibited abscission. Remarkably, depletion of individual ESCRT-III and VPS4 proteins also altered centrosome and spindle pole numbers, producing multipolar spindles (most ESCRT-III/VPS4 proteins) or monopolar spindles (CHMP2A or CHMP5) and causing defects in chromosome segregation and nuclear morphology. VPS4 proteins concentrated at spindle poles during mitosis and then at midbodies during cytokinesis, implying that these proteins function directly at both sites. We conclude that ESCRT-III/VPS4 proteins function at centrosomes to help regulate their maintenance or proliferation and then at midbodies during abscission, thereby helping ensure the ordered progression through the different stages of cell division.

The ubiquitin ligase Nedd4-1 is required for heart development and is a suppressor of thrombospondin-1.

Nedd4 (Nedd4-1) is a Hect domain E3 ubiquitin ligase that also contains a C2 domain and three WW domains. Despite numerous in vitro studies, its biological function in vivo is not well understood. Here we show that disruption of Nedd4-1 in mice (leaving Nedd4-2 intact) caused embryonic lethality at mid gestation, with pronounced heart defects (double-outlet right ventricle and atrioventricular cushion defects) and vasculature abnormalities. Quantitative mass spectrometry and immunoblot analyses of lysates from the wild type and knock-out mouse embryonic fibroblasts to identify Nedd4-1 in vivo targets revealed dramatically increased amounts of thrombospondin-1 (Tsp-1) in the knock-out mouse embryonic fibroblasts and embryos. Tsp-1 is an inhibitor of angiogenesis, and its elevated level was mediated primarily by enhanced transcription. Interestingly, the administration of aspirin (an inhibitor of Tsp-1) to the pregnant heterozygote mothers led to a reduction in Tsp-1 levels and a substantial rescue of the embryonic lethality. These results suggest that Nedd4-1 is a suppressor of Tsp1 and that increased levels of Tsp-1 in the Nedd4-1 knock-out mice may have contributed to the developmental defect observed in the embryos.

Involvement of Snf7p and Rim101p in the transcriptional regulation of TIR1 and other anaerobically upregulated genes in Saccharomyces cerevisiae.

Despite the scientific and applied interest in the anaerobic metabolism of Saccharomyces cerevisiae, not all genes whose transcription is upregulated under anaerobic conditions have yet been linked to known transcription factors. Experiments with a reporter construct in which the promoter of the anaerobically upregulated TIR1 gene was fused to lacZ revealed a loss of anaerobic upregulation in an snf7Delta mutant. Anaerobic upregulation was restored by expression of a truncated allele of RIM101 that encodes for a constitutively active Rim101p. Analysis of lacZ expression in several deletion mutants confirmed that the effect of Snf7p on anaerobic upregulation of TIR1 involved Rim101p. Further studies with deletion mutants in NRG1, NRG2 and SMP1, which were previously shown to be regulated by Rim101p, could not totally elucidate the TIR1 regulation, suggesting the involvement of a more complex regulation network. However, the aerobic repression mechanism of TIR1 involved the general repressor Ssn6p-Tup1p. Transcriptome analysis in anaerobic chemostat cultures revealed that 26 additional genes exhibited an Snf7p/Rim101p-dependent anaerobic upregulation, among which, besides TIR1, are four other anaerobic genes SML1, MUC1, AAC3 and YBR300C. These results provide new evidence on the implication of the Rim101p cascade in the transcriptional regulation of anaerobic metabolism in S. cerevisiae.

ALIX increases protein content and protective function of iPSC-derived exosomes.

Nature of exosome-secreting cells determines exosome content and function. ALIX, involved in exosome biogenesis, promotes cell degeneration. Here, ALIX was knocked out (iPSC-ALIX-/-) and overexpressed (iPSC-ALIX3+) in induced pluripotent stem cells (iPSCs) using CRISPR-Cas9 and lentiviral transduction, respectively, and the secreted exosomes were analyzed. Exosomes from iPSC-ALIX-/- (exosome-KO), iPSC-ALIX3+ (exosome-over), and their corresponding controls contained 176, 529, 431, and 351 proteins, respectively. Exosome-over showed increased protein levels, while exosome-KO contained fewer protein types without differing in total protein content. ALIX knockout did not affect exosome uptake by endothelial cells. Exosome-over more effectively promoted cell viability than exosome-GFP, in a dose-dependent manner. All exosomes were protective for endothelial cells injured by hydrogen peroxide or cisplatin, as demonstrated by promotion of cell viability, horizontal migration, angiogenic sprouting from aortic rings, and formation of capillary-like structures, inhibition of apoptosis, and maintenance of permeability of endothelial monolayer, although exosome-over and exosome-KO had stronger and weaker effects, respectively. SNX2 was important for ALIX-mediated exosomal function. Beneficial functions of the exosomes were independent of experimental models, targeted cell types, causes of injury, exosome-producing iPSC passages, clones of ALIX knockout, and transfection batches of ALIX overexpression. Thus, we present a novel strategy to manipulate iPSCs for production of exosomes with beneficial ALIX-regulated protein composition for varied exosome functions. KEY MESSAGES: ALIX knockout and overexpression regulate protein profile in iPSC-derived exosome. ALIX knockout decreases therapeutic function of iPSC-derived exosomes. ALIX overexpression increases therapeutic function of iPSC-derived exosomes. Manipulating iPSCs can produce exosomes with more beneficial protein content.

Downregulation of LAPTM5 suppresses cell proliferation and viability inducing cell cycle arrest at G0/G1 phase of bladder cancer cells.

Our transcriptome analysis revealed in bladder cancer (BCa) tissues a significant induction of lysosomal-associated multispanning membrane protein 5 (LAPTM5), a lysosomal membrane protein preferentially expressing in immune cells and hematopoietic cells. Transportation of LAPTM5 from Golgi to lysosome could be inhibited by deficiency of Nedd4, a key member of E3 ubiquitin ligase family overexpressing in invasive BCa and promoting its progression. Therefore, we hypothesize that LAPTM5 may be closely correlated with BCa tumorigenesis. In human BCa tissues, we observed that LAPTM5 was significantly induced at both mRNA and protein levels, which is consistent with our microarray result. Furthermore, we established a BCa cell model with downregulated LAPTM5, revealing a significantly delayed growth rate in the BCa cells with knockdown of LAPTM5. Moreover, cell cycle arrest at G0/G1 phase was triggered by decreased LAPTM5 as well, which could lead to delayed BCa cell growth. In contrast, no significant alteration of apoptosis in the BCa cells with downregulated LAPTM5 was noticed. Analysis of the changes of migration and invasion, showed significant reduced LAPTM5 suppressed cell metastasis. Furthermore, proteins involved in epithelial-mesenchymal transition (EMT) were strongly altered, which plays a central role in metastasis. In addition, phosphorylated ERK1/2 and p38, key members of mitogen-activated protein kinase (MAPK) family regulating BCa tumorigenesis, were strongly decreased. Taken together, our results suggested that decreased LAPTM5 inhibited proliferation and viability, as well as induced G0/G1 cell cycle arrest possibly via deactivation of ERK1/2 and p38 in BCa cells.

Intestinal knockout of Nedd4 enhances growth of Apcmin tumors.

Nedd4 (Nedd4-1) is an E3 ubiquitin ligase that belongs to the HECT family and comprises a C2-WW(n)-HECT domain architecture. Although it has been reported to regulate growth factor receptors and cellular signaling, its role in cancer development has been controversial, with some studies proposing that it promotes cancer while others suggest it inhibits tumor growth. Here, we tested the effect of Nedd4 on intestinal tumor formation and growth using Nedd4-knockout mice (Nedd4 floxed (fl) mice crossed to villin-Cre mice). Although we find that knockout of Nedd4 on its own does not cause tumor growth, its knockout in the context of Apc+/min-derived colorectal tumors leads to augmentation of tumor growth, suggesting that Nedd4 normally suppresses intestinal WNT signaling and growth of colonic tumors. WNT signaling microarray, immunoblotting and immunohistochemistry analyses of tumors derived from the Villin-Cre;Nedd4fl/fl;Apc+/min colons demonstrated elevated expression of the WNT upstream effectors LEF1 (full length) and YY1 in these tumors relative to control (Apc+/min alone) tumors. Together, these results suggest that Nedd4 suppresses colonic WNT signaling and tumor growth, at least in part, by suppressing the transcription factors LEF1 and YY1.

The Legionella pneumophila effector protein, LegC7, alters yeast endosomal trafficking.

The intracellular pathogen, Legionella pneumophila, relies on numerous secreted effector proteins to manipulate host endomembrane trafficking events during pathogenesis, thereby preventing fusion of the bacteria-laden phagosome with host endolysosomal compartments, and thus escaping degradation. Upon expression in the surrogate eukaryotic model Saccharomyces cerevisiae, we find that the L. pneumophila LegC7/YlfA effector protein disrupts the delivery of both biosynthetic and endocytic cargo to the yeast vacuole. We demonstrate that the effects of LegC7 are specific to the endosome:vacuole delivery pathways; LegC7 expression does not disrupt other known vacuole-directed pathways. Deletions of the ESCRT-0 complex member, VPS27, provide resistance to the LegC7 toxicity, providing a possible target for LegC7 function in vivo. Furthermore, a single amino acid substitution in LegC7 abrogates both its toxicity and ability to alter endosomal traffic in vivo, thereby identifying a critical functional domain. LegC7 likely inhibits endosomal trafficking during L. pneumophila pathogenesis to prevent entry of the phagosome into the endosomal maturation pathway and eventual fusion with the lysosome.

Smooth Muscle Hgs Deficiency Leads to Impaired Esophageal Motility.

As a master component of endosomal sorting complex required for transport proteins, hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs) participates multiple cellular behaviors. However, the physiological role of Hgs in smooth muscle cells (SMCs) is by far unknown. Here we explored the in vivo function of Hgs in SMCs by using a conditional gene knockout strategy. Hgs deficiency in SMCs uniquely led to a progressive dilatation of esophagus with a remarkable thinning muscle layer. Of note, the mutant esophagus showed a decreased contractile responsiveness to potassium chloride and acetylcholine stimulation. Furthermore, an increase in the inhibitory neurites along with an intense infiltration of T lymphocytes in the mucosa and muscle layer were observed. Consistently, Hgs deficiency in SMCs resulted in a disturbed expression of a set of genes involved in neurotrophin and inflammation, suggesting that defective SMC might be a novel source for excessive production of cytokines and chemokines which may trigger the neuronal dysplasia and ultimately contribute to the compromised esophageal motility. The data suggest potential implications in the pathogenesis of related diseases such as gastroesophageal reflux disease.