Caenorhabditis elegans PTR/PTCHD PTR-18 promotes the clearance of extracellular hedgehog-related protein via endocytosis.

Spatiotemporal restriction of signaling plays a critical role in animal development and tissue homeostasis. All stem and progenitor cells in newly hatched C. elegans larvae are quiescent and capable of suspending their development until sufficient food is supplied. Here, we show that ptr-18, which encodes the evolutionarily conserved patched-related (PTR)/patched domain-containing (PTCHD) protein, temporally restricts the availability of extracellular hedgehog-related protein to establish the capacity of progenitor cells to maintain quiescence. We found that neural progenitor cells exit from quiescence in ptr-18 mutant larvae even when hatched under starved conditions. This unwanted reactivation depended on the activity of a specific set of hedgehog-related grl genes including grl-7. Unexpectedly, neither PTR-18 nor GRL-7 were expressed in newly hatched wild-type larvae. Instead, at the late embryonic stage, both PTR-18 and GRL-7 proteins were first localized around the apical membrane of hypodermal and neural progenitor cells and subsequently targeted for lysosomal degradation before hatching. Loss of ptr-18 caused a significant delay in GRL-7 clearance, causing this protein to be retained in the extracellular space in newly hatched ptr-18 mutant larvae. Furthermore, the putative transporter activity of PTR-18 was shown to be required for the appropriate function of the protein. These findings not only uncover a previously undescribed role of PTR/PTCHD in the clearance of extracellular hedgehog-related proteins via endocytosis-mediated degradation but also illustrate that failure to temporally restrict intercellular signaling during embryogenesis can subsequently compromise post-embryonic progenitor cell function.

Development of a versatile HPLC-based method to evaluate the activation status of small GTPases.

Small GTPases cycle between an inactive GDP-bound and an active GTP-bound state to control various cellular events, such as cell proliferation, cytoskeleton organization, and membrane trafficking. Clarifying the guanine nucleotide-bound states of small GTPases is vital for understanding the regulation of small GTPase functions and the subsequent cellular responses. Although several methods have been developed to analyze small GTPase activities, our knowledge of the activities for many small GTPases is limited, partly because of the lack of versatile methods to estimate small GTPase activity without unique probes and specialized equipment. In the present study, we developed a versatile and straightforward HPLC-based assay to analyze the activation status of small GTPases by directly quantifying the amounts of guanine nucleotides bound to them. This assay was validated by analyzing the RAS-subfamily GTPases, including HRAS, which showed that the ratios of GTP-bound forms were comparable with those obtained in previous studies. Furthermore, we applied this assay to the investigation of psychiatric disorder-associated mutations of RHEB (RHEB/P37L and RHEB/S68P), revealing that both mutations cause an increase in the ratio of the GTP-bound form in cells. Mechanistically, loss of sensitivity to TSC2 (a GTPase-activating protein for RHEB) for RHEB/P37L, as well as both decreased sensitivity to TSC2 and accelerated guanine-nucleotide exchange for RHEB/S68P, is involved in the increase of their GTP-bound forms, respectively. In summary, the HPLC-based assay developed in this study provides a valuable tool for analyzing small GTPases for which the activities and regulatory mechanisms are less well understood.

epi-Aszonalenin B from Aspergillus novofumigatus inhibits NF-κB activity induced by ZFTA-RELA fusion protein that drives ependymoma.

Nuclear factor-kappa B (NF-κB) signaling is an intracellular signaling pathway involved in inflammatory responses and the pathogenesis of various cancers, including ependymoma, which is a rare and chemotherapy-resistant glioma. Several isoforms of fusion proteins that consist of a nuclear protein, zinc finger translocation associated (ZFTA), and RELA (ZFTA-RELA), an NF-κB-signaling effector transcription factor, cause excessive activation of the NF-κB signaling pathway and result in supratentorial ependymomas (ST-EPN-RELA). As inhibitors of NF-κB activity induced by ZFTA-RELA are expected to be therapeutic agents for ST-EPN-RELA, we established an NF-κB responsive luciferase reporter cell line that expresses the most common isoform of ZFTA-RELA in a doxycycline-dependent manner. Using this reporter cell line, we screened fungus extracts for compounds that inhibit the NF-κB activity induced by ZFTA-RELA expression and identified aszonalenin, an alkaloid from Aspergillus novofumigatus. We also purified analogs of aszonalenin, namely acetylaszonalenin and epi-aszonalenin B and C. In a luciferase assay using cells constitutively expressing luciferase (counter assay), acetylaszonalenin and epi-aszonalenin C showed non-specific inhibition of the luciferase activity. Aszonalenin and epi-aszonalenin B inhibited the NF-κB responsive luciferase activity by expressing ZFTA-RELA more strongly than the luciferase activity in the counter assay. The upregulation of endogenous NF-κB responsive genes, such as CCND1, ICAM1, and L1CAM, by ZFTA-RELA expression was inhibited by epi-aszonalenin B, but not by aszonalenin. This study suggests that epi-aszonalenin B may be a lead compound for the therapeutic development of ST-EPN-RELA.

GEF mechanism revealed by the structure of SmgGDS-558 and farnesylated RhoA complex and its implication for a chaperone mechanism.

SmgGDS has dual functions in cells and regulates small GTPases as both a guanine nucleotide exchange factor (GEF) for the Rho family and a molecular chaperone for small GTPases possessing a C-terminal polybasic region followed by four C-terminal residues called the CaaX motif, which is posttranslationally prenylated at its cysteine residue. Our recent structural work revealed that SmgGDS folds into tandem copies of armadillo-repeat motifs (ARMs) that are not present in other GEFs. However, the precise mechanism of GEF activity and recognition mechanism for the prenylated CaaX motif remain unknown because SmgGDS does not have a typical GEF catalytic domain and lacks a pocket to accommodate a prenyl group. Here, we aimed to determine the crystal structure of the SmgGDS/farnesylated RhoA complex. We found that SmgGDS induces a significant conformational change in the switch I and II regions that opens up the nucleotide-binding site, with the prenyl group fitting into the cryptic pocket in the N-terminal ARMs. Taken together, our findings could advance the understanding of the role of SmgGDS and enable drug design strategies for targeting SmgGDS and small GTPases.

Loss of the small GTPase Arl8b results in abnormal development of the roof plate in mouse embryos.

Lysosomes are acidic organelles responsible for degrading both exogenous and endogenous materials. The small GTPase Arl8 localizes primarily to lysosomes and is involved in lysosomal function. In the present study, using Arl8b gene-trapped mutant (Arl8b-/- ) mice, we show that Arl8b is required for the development of dorsal structures of the neural tube, including the thalamus and hippocampus. In embryonic day (E) 10.5 Arl8b-/- embryos, Sox1 (a neuroepithelium marker) was ectopically expressed in the roof plate, whereas the expression of Gdf7 and Msx1 (roof plate markers) was reduced in the dorsal midline of the midbrain. Ectopic expression of Sox1 in Arl8b-/- embryos was detected also at E9.0 in the neural fold, which gives rise to the roof plate. In addition, the levels of Bmp receptor IA and phosphorylated Smad 1/5/8 (downstream of BMP signaling) were increased in the neural fold of E9.0 Arl8b-/- embryos. These results suggest that Arl8b is involved in the development of the neural fold and the subsequently formed roof plate, possibly via control of BMP signaling.

Arl8b is required for lysosomal degradation of maternal proteins in the visceral yolk sac endoderm of mouse embryos.

The small GTPase Arl8b localizes primarily to lysosomes and is involved in lysosomal motility and fusion. Here, we show that Arl8b is required for lysosomal degradation of maternal proteins in the visceral yolk sac endoderm (VYSE), an apical cell layer of the visceral yolk sac, of mouse embryos. The VYSE actively takes up maternal materials from uterine fluid and degrades them in lysosomes to provide breakdown products to the embryo. Arl8b gene-trap mice (Arl8b-/- ) displayed decreased early embryo body size. The Arl8b-/-  VYSE exhibited defective endocytic trafficking to the lysosome and accumulation of maternal proteins such as albumin and immunoglobulin G in late endocytic organelles. Furthermore, Transthyretin-Cre;Arl8bflox/flox mice in which Arl8b was ablated specifically in the VYSE also showed decreased embryo body size, defects in trafficking to the lysosome and reduction of the free amino acid level in the embryos. Taken together, these results suggest that Arl8b mediates lysosomal degradation of maternal proteins in the VYSE, thereby contributing to mouse embryonic development.

Hedgehog-related genes regulate reactivation of quiescent neural progenitors in Caenorhabditis elegans.

The animal body contains various types of stem and progenitor cells. These undifferentiated cells coordinate the balance between quiescence and proliferation with dynamics of various physiological conditions such as the developmental stage, food availability, and injury. Although regulation of such coordination plays a critical role in maintaining tissue homeostasis, controlling the growth rate and regeneration, much of its mechanism remains elusive. Newly hatched Caenorhabditis elegans larvae possess quiescent stem and progenitor cells in several tissues, and these cells are reactivated by the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway only when sufficient food is supplied. Maintenance of the quiescence of neuronal and mesodermal progenitor cells requires microRNA (miRNA), miR-235, which is upregulated under the starvation. On the other hand, feeding ample food downregulates the miRNA via the activity of the IIS pathway. As miR-235 in the hypodermis can non-autonomously regulate quiescence of neuronal and mesodermal progenitor cells, a cell-cell signaling pathway has been hypothesized to act downstream of the miRNA. Here, we provide evidence that two hedgehog-related (hh-r) genes, grl-5 and grl-7, are targets of miR-235 that promote reactivation of quiescent neuroblasts. These grl genes possess an miR-235 binding site on 3'UTRs of their transcripts, and are upregulated in starved mir-235 mutant larvae. grl-5 and grl-7 promoters can continuously drive the expression of GFP-pest reporter protein in the hypodermis under the fed condition. However, expression of these reporters is strikingly downregulated under the starvation condition after hatching. We found that miR-235 can repress expression of reporter genes via the predicted miR-235 binding sites on the grl-5 and grl-7 3'UTRs. Furthermore, activity of grl-5 and grl-7 genes are required for reactivation of neural progenitor cells in starved mir-235 mutant larvae. These findings suggest that the IIS pathway-miR-235 signaling in the hypodermis non-autonomously regulates quiescence of neural progenitor cells, partly via grl-5 and grl-7.

The microRNA miR-235 couples blast-cell quiescence to the nutritional state.

The coordination of stem- and blast-cell behaviours, such as self-renewal, differentiation and quiescence, with physiological changes underlies growth, regeneration and tissue homeostasis. Germline stem and somatic blast cells in newly hatched Caenorhabditis elegans larvae can suspend postembryonic development, which consists of diverse cellular events such as migration, proliferation and differentiation, until the nutritional state becomes favourable (termed L1 diapause). Although previous studies showed that the insulin/insulin-like growth factor (IGF) signalling (IIS) pathway regulates this developmental quiescence, the detailed mechanism by which the IIS pathway enables these multipotent cells to respond to nutrient availability is unknown. Here we show in C. elegans that the microRNA (miRNA) miR-235, a sole orthologue of mammalian miR-92 from the oncogenic miR-17-92 cluster, acts in the hypodermis and glial cells to arrest postembryonic developmental events in both neuroblasts and mesoblasts. Expression of mir-235 persists during L1 diapause, and decreases upon feeding in a manner dependent on the IIS pathway. Upregulation of one of the miR-235 targets, nhr-91, which encodes an orthologue of mammalian germ cell nuclear factor, is responsible for defects caused by loss of the miRNA. Our findings establish a novel role of a miR-92 orthologue in coupling blast-cell behaviours to the nutritional state.

Structure-based analysis of the guanine nucleotide exchange factor SmgGDS reveals armadillo-repeat motifs and key regions for activity and GTPase binding.

Small GTPases are molecular switches that have critical biological roles and are controlled by GTPase-activating proteins and guanine nucleotide exchange factors (GEFs). The smg GDP dissociation stimulator (SmgGDS) protein functions as a GEF for the RhoA and RhoC small GTPases. SmgGDS has various regulatory roles, including small GTPase trafficking and localization and as a molecular chaperone, and interacts with many small GTPases possessing polybasic regions. Two SmgGDS splice variants, SmgGDS-558 and SmgGDS-607, differ in GEF activity and binding affinity for RhoA depending on the lipidation state, but the reasons for these differences are unclear. Here we determined the crystal structure of SmgGDS-558, revealing a fold containing tandem copies of armadillo repeats not present in other GEFs. We also observed that SmgGDS harbors distinct positively and negatively charged regions, both of which play critical roles in binding to RhoA and GEF activity. This is the first report demonstrating a relationship between the molecular function and atomic structure of SmgGDS. Our findings indicate that the two SmgGDS isoforms differ in GTPase binding and GEF activity, depending on the lipidation state, thus providing useful information about the cellular functions of SmgGDS in cells.

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord.

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord.

Variegated RHOA mutations in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers.

Di-Ras2 Protein Forms a Complex with SmgGDS Protein in Brain Cytosol in Order to Be in a Low Affinity State for Guanine Nucleotides.

The Ras family of small GTPases function in a wide variety of biological processes as "molecular switches" by cycling between inactive GDP-bound and active GTP-bound forms. Di-Ras1 and Di-Ras2 were originally identified as small GTPases forming a distinct subgroup of the Ras family. Di-Ras1/Di-Ras2 mRNAs are detected predominantly in brain and heart tissues. Biochemical analysis of Di-Ras1/Di-Ras2 has revealed that they have little GTPase activity and that their intrinsic guanine-nucleotide exchange rates are much faster than that of H-Ras. Yet little is known about the biological role(s) of Di-Ras1/Di-Ras2 or of how their activities are regulated. In the present study we found that endogenous Di-Ras2 co-purifies with SmgGDS from rat brain cytosol. Size-exclusion chromatography of purified recombinant proteins showed that Di-Ras2 forms a high affinity complex with SmgGDS. SmgGDS is a guanine nucleotide exchange factor with multiple armadillo repeats and has recently been shown to specifically activate RhoA and RhoC. In contrast to the effect on RhoA, SmgGDS does not act as a guanine nucleotide exchange factor for Di-Ras2 but instead tightly associates with Di-Ras2 to reduce its binding affinity for guanine nucleotides. Finally, pulse-chase analysis revealed that Di-Ras2 binds, in a C-terminal CAAX motif-dependent manner, to SmgGDS immediately after its synthesis. This leads to increased Di-Ras2 stability. We thus propose that isoprenylated Di-Ras2 forms a tight complex with SmgGDS in cytosol immediately after its synthesis, which lowers its affinity for guanine nucleotides.

Genetically encoded calcium indicator illuminates calcium dynamics in primary cilia.

Visualization of signal transduction in live primary cilia constitutes a technical challenge owing to the organelle's submicrometer dimensions and close proximity to the cell body. Using a genetically encoded calcium indicator targeted to primary cilia, we visualized calcium signaling in cilia of mouse fibroblasts and kidney cells upon chemical or mechanical stimulation with high specificity, high sensitivity and wide dynamic range.

Mechanisms for exporting large-sized cargoes from the endoplasmic reticulum.

Cargo proteins exported from the endoplasmic reticulum to the Golgi apparatus are typically transported in coat protein complex II (COPII)-coated vesicles of 60-90 nm diameter. Several cargo molecules including collagens and chylomicrons form structures that are too large to be accommodated by these vesicles, but their secretion still requires COPII proteins. Here, we first review recent progress on large cargo secretions derived especially from animal models and human diseases, which indicate the importance of COPII proteins. We then discuss the recent isolation of specialized factors that modulate the process of COPII-dependent cargo formation to facilitate the exit of large-sized cargoes from the endoplasmic reticulum. Based on these findings, we propose a model that describes the importance of the GTPase cycle for secretion of oversized cargoes. Next, we summarize reports that describe the structures of COPII proteins and how these results provide insight into the mechanism of assembly of the large cargo carriers. Finally, we discuss what issues remain to be solved in the future.

Transforming mutations of RAC guanosine triphosphatases in human cancers.

Members of the RAS superfamily of small guanosine triphosphatases (GTPases) transition between GDP-bound, inactive and GTP-bound, active states and thereby function as binary switches in the regulation of various cellular activities. Whereas HRAS, NRAS, and KRAS frequently acquire transforming missense mutations in human cancer, little is known of the oncogenic roles of other small GTPases, including Ras-related C3 botulinum toxin substrate (RAC) proteins. We show that the human sarcoma cell line HT1080 harbors both NRAS(Q61K) and RAC1(N92I) mutant proteins. Whereas both of these mutants were able to transform fibroblasts, knockdown experiments indicated that RAC1(N92I) may be the essential growth driver for this cell line. Screening for RAC1, RAC2, or RAC3 mutations in cell lines and public databases identified several missense mutations for RAC1 and RAC2, with some of the mutant proteins, including RAC1(P29S), RAC1(C157Y), RAC2(P29L), and RAC2(P29Q), being found to be activated and transforming. P29S, N92I, and C157Y mutants of RAC1 were shown to exist preferentially in the GTP-bound state as a result of a rapid transition from the GDP-bound state, rather than as a result of a reduced intrinsic GTPase activity. Activating mutations of RAC GTPases were thus found in a wide variety of human cancers at a low frequency; however, given their marked transforming ability, the mutant proteins are potential targets for the development of new therapeutic agents.

Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine.

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

Active transport and diffusion barriers restrict Joubert Syndrome-associated ARL13B/ARL-13 to an Inv-like ciliary membrane subdomain.

Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.

Biological role of the two overlapping poly(A)-binding protein interacting motifs 2 (PAM2) of eukaryotic releasing factor eRF3 in mRNA decay.

Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1-Ccr4 and Pan2-Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2-PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 3' end of poly(A).

Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence.

The development of antifungal drugs requires novel molecular targets due to limited treatment options and drug resistance. Through chemical screening and establishment of a novel genetic technique to repress gene expression in Trichophyton rubrum, the primary causal fungus of dermatophytosis, we demonstrated that fungal Cdc42 and Rac GTPases are promising antifungal drug targets. Chemical inhibitors of these GTPases impair hyphal formation, which is crucial for growth and virulence in T. rubrum. Conditional repression of Cdc24, a guanine nucleotide exchange factor for Cdc42 and Rac, led to hyphal growth defects, abnormal cell morphology, and cell death. EHop-016 inhibited the promotion of the guanine nucleotide exchange reaction in Cdc42 and Rac by Cdc24 as well as germination and growth on the nail fragments of T. rubrum and improved animal survival in an invertebrate infection model of T. rubrum. Our results provide a novel antifungal therapeutic target and a potential lead compound.

A novel acetylation cycle of transcription co-activator Yes-associated protein that is downstream of Hippo pathway is triggered in response to SN2 alkylating agents.

Yes-associated protein (YAP) is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes. Although cytoplasmic retention of YAP is known to be mediated by Hippo pathway-dependent phosphorylation, post-translational modifications that regulate YAP in the nucleus remain unclear. Here we report the discovery of a novel cycle of acetylation/deacetylation of nuclear YAP induced in response to S(N)2 alkylating agents. We show that after treatment of cells with the S(N)2 alkylating agent methyl methanesulfonate, YAP phosphorylation mediated by the Hippo pathway is markedly reduced, leading to nuclear translocation of YAP and its acetylation. This YAP acetylation occurs on specific and highly conserved C-terminal lysine residues and is mediated by the nuclear acetyltransferases CBP (CREB binding protein) and p300. Conversely, the nuclear deacetylase SIRT1 is responsible for YAP deacetylation. Intriguingly, we found that YAP acetylation is induced specifically by S(N)2 alkylating agents and not by other DNA-damaging stimuli. These results identify a novel YAP acetylation cycle that occurs in the nucleus downstream of the Hippo pathway. Intriguingly, our findings also indicate that YAP acetylation is involved in responses to a specific type of DNA damage.