Search

Crohn's disease (CD) is a complex and heterogeneous condition with no perfect preclinical model or cure. To address this, we explore adult stem cell-derived organoids that retain their tissue identity and disease-driving traits. We prospectively create a biobank of CD patient-derived organoid cultures (PDOs) from colonic biopsies of 53 subjects across all clinical subtypes and healthy subjects. Gene expression analyses enabled benchmarking of PDOs as tools for modeling the colonic epithelium in active disease and identified two major molecular subtypes: immune-deficient infectious CD (IDICD) and stress and senescence-induced fibrostenotic CD (S2FCD). Each subtype shows internal consistency in the transcriptome, genome, and phenome. The spectrum of morphometric, phenotypic, and functional changes within the "living biobank" reveals distinct differences between the molecular subtypes. Drug screens reverse subtype-specific phenotypes, suggesting phenotyped-genotyped CD PDOs can bridge basic biology and patient trials by enabling preclinical phase "0" human trials for personalized therapeutics., Competing Interests: Declaration of interests S.D. and P.G. have a patent on the methodology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Trichomonas vaginalis (Tv) is a parasite that causes trichomoniasis, a prevalent sexually-transmitted infection. Neutrophils are found at the site of infection, and can rapidly kill the parasite in vitro, using trogocytosis. However, the specific molecular players in neutrophil killing of Tv are unknown. Here, we show that complement proteins play a role in Tv killing by human neutrophil-like cells (NLCs). Using CRISPR/Cas9, we generated NLCs deficient in each of three complement receptors (CRs) known to be expressed on human neutrophils: CR1, CR3, and CR4. Using in vitro trogocytosis assays, we found that CR3, but not CR1 or CR4 is required for maximum trogocytosis of the parasite by NLCs, with NLCs lacking CR3 demonstrating ~40% reduction in trogocytosis, on average. We also observed a reduction in NLC killing of Tv in CR3 knockout, but not CR1 or CR4 knockout NLCs. On average, NLCs lacking CR3 had ~50% reduction in killing activity. We also used a parallel approach of pre-incubating NLCs with blocking antibodies against CR3, which similarly reduced NLC killing of parasites. These data support a model in which Tv is opsonized by the complement protein iC3b, and bound by neutrophil CR3 receptor, to facilitate trogocytic killing of the parasite., (© 2024 The Authors. Parasite Immunology published by John Wiley & Sons Ltd.)

Crohn's disease (CD) is a complex, clinically heterogeneous disease of multifactorial origin; there is no perfect pre-clinical model, little insight into the basis for such heterogeneity, and still no cure. To address these unmet needs, we sought to explore the translational potential of adult stem cell-derived organoids that not only retain their tissue identity, but also their genetic and epigenetic disease-driving traits. We prospectively created a biobank of CD patient-derived organoid cultures (PDOs) using biopsied tissues from colons of 34 consecutive subjects representing all clinical subtypes (Montreal Classification B1-B3 and perianal disease). PDOs were generated also from healthy subjects. Comparative gene expression analyses enabled benchmarking of PDOs as tools for modeling the colonic epithelium in active disease and revealed that despite the clinical heterogeneity there are two major molecular subtypes: immune-deficient infectious-CD [IDICD] and stress and senescence-induced fibrostenotic-CD [S2FCD]. The transcriptome, genome and phenome show a surprising degree of internal consistency within each molecular subtype. The spectrum of morphometric, phenotypic, and functional changes within the "living biobank" reveals distinct differences between the molecular subtypes. These insights enabled drug screens that reversed subtype-specific phenotypes, e.g., impaired microbial clearance in IDICD was reversed using agonists for nuclear receptors, and senescence in S2FCD was rectified using senotherapeutics, but not vice versa . Phenotyped-genotyped CD-PDOs may fill the gap between basic biology and patient trials by enabling pre-clinical Phase '0' human trials for personalized therapeutics., In Brief: This work creates a prospectively biobanked phenotyped-genotyped Crohn's disease patient-derived organoids (CD-PDOs) as platforms for molecular subtyping of disease and for ushering personalized therapeutics., Highlights: Prospectively biobanked CD-organoids recapitulate the disease epithelium in patientsThe phenome-transcriptome-genome of CD-organoids converge on two molecular subtypesOne subtype shows impaired microbial clearance, another increased cellular senescencePhenotyped-genotyped PDOs are then used for integrative and personalized therapeutics.

Upon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of the two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DSBs hinges on GIV/Girdin, a guanine nucleotide-exchange modulator of heterotrimeric Giα•βγ protein. GIV suppresses HR by binding and sequestering BRCA1, a key coordinator of multiple steps within the HR pathway, away from DSBs; it does so using a C-terminal motif that binds BRCA1's BRCT-modules via both phospho-dependent and -independent mechanisms. Using another non-overlapping C-terminal motif GIV binds and activates Gi and enhances the "free" Gβγ→PI-3-kinase→Akt pathway, which promotes survival and is known to suppress HR, favor NHEJ. Absence of GIV, or loss of either of its C-terminal motifs enhanced cell death upon genotoxic stress. Because GIV selectively binds other BRCT-containing proteins suggests that G-proteins may fine-tune sensing, repair, and survival after diverse types of DNA damage., Competing Interests: Authors declare no competing interests., (© 2023 The Author(s).)

E-cigarette usage continues to rise, yet the safety of e-cigarette aerosols is questioned. Using murine models of acute and chronic e-cigarette aerosol inhalation, murine colon transcriptomics, and murine and human gut-derived organoids in co-culture models, we assessed the effects of e-cigarette use on the gut barrier. Histologic and transcriptome analyses revealed that chronic, but not acute, nicotine-free e-cigarette use increased inflammation and reduced expression of tight junction (TJ) markers. Exposure of murine and human enteroid-derived monolayers (EDMs) to nicotine-free e-cigarette aerosols alone or in co-culture with bacteria also causes barrier disruption, downregulation of TJ protein, and enhanced inflammation in response to infection. These data highlight the harmful effects of "non-nicotine" component of e-cigarettes on the gut barrier. Considering the importance of an intact gut barrier for host fitness and the impact of gut mucosal inflammation on a multitude of chronic diseases, these findings are broadly relevant to both medicine and public health., Competing Interests: S.D. and P.G. have patents on methodology to prepare enteroid monolayers and functional assays related to the gut barrier. The authors have declared that no other conflict of interest exists., (© 2021 The Author(s).)

PDZ domains are one of the most abundant protein domains in eukaryotes and are frequently found on junction-localized scaffold proteins. Various signaling molecules bind to PDZ proteins via PDZ-binding motifs (PBM) and fine-tune cellular signaling. However, how such interaction affects protein function is difficult to predict and must be solved empirically. Here we describe a long isoform of the guanine nucleotide exchange factor GIV/Girdin (CCDC88A) that we named GIV-L, which is conserved throughout evolution, from invertebrates to vertebrates, and contains a PBM. Unlike GIV, which lacks PBM and is cytosolic, GIV-L localizes onto cell junctions and has a PDZ interactome (as shown through annotating Human Cell Map and BioID-proximity labeling studies), which impacts GIV-L's ability to bind and activate trimeric G-protein, Gαi, through its guanine-nucleotide exchange modulator (GEM) module. This GEM module is found exclusively in vertebrates. We propose that the two functional modules in GIV may have evolved sequentially: the ability to bind PDZ proteins via the PBM evolved earlier in invertebrates, whereas G-protein binding and activation may have evolved later only among vertebrates. Phenotypic studies in Caco-2 cells revealed that GIV and GIV-L may have antagonistic effects on cell growth, proliferation (cell cycle), and survival. Immunohistochemical analysis in human colon tissues showed that GIV expression increases with a concomitant decrease in GIV-L during cancer initiation. Taken together, these findings reveal how regulation in GIV/CCDC88A transcript helps to achieve protein modularity, which allows the protein to play opposing roles either as a tumor suppressor (GIV-L) or as an oncogene (GIV)., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Besides being regulated by G-protein-coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE ( D vl- a ssociating p rotein with a high frequency of le ucine) are the best-characterized members of a group of cytoplasmic regulators that contain a Gα-binding and -activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif-containing proteins were originally reported to modulate G proteins by binding Gα subunits of the G i/o family (Gα i ) over other families (such as G s , G q/11 , or G 12/13 ), and promoting nucleotide exchange in vitro However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gα s and inhibits nucleotide exchange. The G-protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gα i remain to be investigated. Here, we show that DAPLE's GBA motif, in addition to Gα i , binds efficiently to members of the G s and G q/11 families (Gα s and Gα q , respectively), but not of the G 12/13 family (Gα 12 ) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gα s and Gα q Unlike the nucleotide-exchange acceleration observed for Gα i , DAPLE inhibited nucleotide exchange on Gα s and Gα q These findings indicate that GBA motifs have versatility in their G-protein-modulating effect, i.e. they can bind to Gα subunits of different classes and either stimulate or inhibit nucleotide exchange depending on the G-protein subtype., (© 2020 Marivin et al.)

Polarized distribution of organelles and molecules inside a cell is vital for a range of cellular processes and its loss is frequently encountered in disease. Polarization during planar cell migration is a special condition in which cellular orientation is triggered by cell-cell contact. We demonstrate that the protein Daple (CCDC88C) is a component of cell junctions in epithelial cells which serves like a cellular "compass" for establishing and maintaining contact-triggered planar polarity. Furthermore, these processes may be mediated through interaction with the polarity regulator PARD3. This interaction, mediated by Daple's PDZ-binding motif (PBM) and the third PDZ domain of PARD3, is fine-tuned by tyrosine phosphorylation on Daple's PBM by receptor and non-receptor tyrosine kinases, such as Src. Hypophosphorylation strengthens the interaction, whereas hyperphosphorylation disrupts it, thereby revealing an unexpected role of Daple as a platform for signal integration and gradient sensing for tyrosine-based signals within the planar cell polarity pathway., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Previously, Aznar et al., showed that Daple/CCDC88C enables Wnt receptors to transactivate trimeric G-proteins during non-canonical Wnt signaling via a novel G-protein binding and activating (GBA) motif. By doing so, Daple serves two opposing roles; earlier during oncogenesis it suppresses neoplastic transformation and tumor growth, but later it triggers epithelial-to-mesenchymal-transition (EMT). We have identified and characterized two isoforms of the human Daple gene. While both isoforms cooperatively suppress tumor growth via their GBA motif, only the full-length transcript triggers EMT and invasion. Both isoforms are suppressed during colon cancer progression, and their reduced expression carries additive prognostic significance. These findings provide insights into the opposing roles of Daple during cancer progression and define the G-protein regulatory GBA motif as one of the minimal modules essential for Daple's role as a tumor suppressor.

A loss of balance between G protein activation and deactivation has been implicated in the initiation of melanomas, and non-canonical Wnt signaling via the Wnt5A/Frizzled (FZD) pathway has been shown to be critical for the switch to an invasive phenotype. Daple [CCDC88C], a cytosolic guanine nucleotide exchange modulator (GEM) which enhances non-canonical Wnt5A/FZD signaling via activation of trimeric G protein, Gαi, has been shown to serve opposing roles-as an inducer of EMT and invasiveness and a potent tumor suppressor-via two isoforms, V1 (full-length) and V2 (short spliced isoform), respectively. Here we report that the relative abundance of these isoforms in the peripheral circulation, presumably largely from circulating tumor cells (CTCs), is a prognostic marker of cutaneous melanomas. Expression of V1 is increased in both the early and late clinical stages (p < 0.001, p = 0.002, respectively); V2 is decreased exclusively in the late clinical stage (p = 0.003). The two isoforms have opposing prognostic effects: high expression of V2 increases relapse-free survival (RFS; p = 0.014), whereas high expression of V1 tends to decrease RFS (p = 0.051). Furthermore, these effects are additive, in that melanoma patients with a low V2-high V1 signature carry the highest risk of metastatic disease. We conclude that detection of Daple transcripts in the peripheral blood (i.e., liquid biopsies) of patients with melanoma may serve as a prognostic marker and an effective strategy for non-invasive long-term follow-up of patients with melanoma.

Cellular proliferation, differentiation, and morphogenesis are shaped by multiple signaling cascades, and their dysregulation plays an integral role in cancer progression. Three cascades that contribute to oncogenic potential are those mediated by Wnt proteins and the receptor Frizzled (FZD), growth factor receptor tyrosine kinases (RTKs), and heterotrimeric G proteins and associated GPCRs. Daple is a guanine nucleotide exchange factor (GEF) for the G protein G αi Daple also binds to FZD and the Wnt/FZD mediator Dishevelled (Dvl), and it enhances β-catenin-independent Wnt signaling in response to Wnt5a-FZD7 signaling. We identified Daple as a substrate of multiple RTKs and non-RTKs and, hence, as a point of convergence for the three cascades. We found that phosphorylation near the Dvl-binding motif in Daple by both RTKs and non-RTKs caused Daple/Dvl complex dissociation and augmented the ability of Daple to bind to and activate G αi , which potentiated β-catenin-independent Wnt signals and stimulated epithelial-mesenchymal transition (EMT) similarly to Wnt5a/FZD7 signaling. Although Daple acts as a tumor suppressor in the healthy colon, the concurrent increased abundance of Daple and epidermal growth factor receptor (EGFR) in colorectal tumors was associated with poor patient prognosis. Thus, the Daple-dependent activation of G αi and the Daple-dependent enhancement of β-catenin-independent Wnt signals are not only stimulated by Wnt5a/FZD7 to suppress tumorigenesis but also hijacked by growth factor-activated RTKs to enhance tumor progression. These findings identify a cross-talk paradigm among growth factor RTKs, heterotrimeric G proteins, and the Wnt/FZD pathway in cancer., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cellular proliferation is antagonistically regulated by canonical and noncanonical Wnt signals; their dysbalance triggers cancers. We previously showed that a multimodular signal transducer, Daple, enhances PI3-K→Akt signals within the noncanonical Wnt signaling pathway and antagonistically inhibits canonical Wnt responses. Here we demonstrate that the PI3-K→Akt pathway serves as a positive feedback loop that further enhances noncanonical Wnt signals by compartmentalizing β-catenin. By phosphorylating the phosphoinositide- (PI) binding domain of Daple, Akt abolishes Daple's ability to bind PI3-P-enriched endosomes that engage dynein motor complex for long-distance trafficking of β-catenin/E-cadherin complexes to pericentriolar recycling endosomes (PCREs). Phosphorylation compartmentalizes Daple/β-catenin/E-cadherin complexes to cell-cell contact sites, enhances noncanonical Wnt signals, and thereby suppresses colony growth. Dephosphorylation compartmentalizes β-catenin on PCREs, a specialized compartment for prolonged unopposed canonical Wnt signaling, and enhances colony growth. Cancer-associated Daple mutants that are insensitive to Akt mimic a constitutively dephosphorylated state. This work not only identifies Daple as a platform for cross-talk between Akt and the noncanonical Wnt pathway but also reveals the impact of such cross-talk on tumor cell phenotypes that are critical for cancer initiation and progression., (© 2017 Aznar et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features. As in the case of any new pathway/paradigm, these studies first required an in-depth optimization of tools/techniques and protocols, governed by rationale and fundamentals unique to the pathway, and more specifically to the large multimodular GIV protein. Here we provide the most up-to-date overview of protocols that have generated most of what we know today about noncanonical G protein activation by GIV and its relevance in health and disease. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK-ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV-Gαi complexes and activates GIV's GEF function; then a second phosphomodification terminates GIV's GEF function, triggers the assembly of GIV-Gαs complexes, and activates GIV's GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK-ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses., Competing Interests: The authors declare no conflict of interest.

5q-syndrome is a distinct form of myelodysplastic syndrome (MDS) where a deletion on chromosome 5 is the underlying cause. MDS is characterized by bone marrow failures, including macrocytic anemia. Genetic mapping and studies using various models support the notion that ribosomal protein S14 (RPS14) is the candidate gene for the erythroid failure. Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis, similar to what is observed with other ribosomal proteins. However, due to the higher risk for cancer development in patients with ribosome deficiency, targeting the p53 pathway is not a viable treatment option. To better understand the pathology of RPS14 deficiency in 5q-deletion, we generated a zebrafish model harboring a mutation in the RPS14 gene. This model mirrors the anemic phenotype seen in 5q-syndrome. Moreover, the anemia is due to a late-stage erythropoietic defect, where the erythropoietic defect is initially p53-independent and then becomes p53-dependent. Finally, we demonstrate the versatility of this model to test various pharmacological agents, such as RAP-011, L-leucine, and dexamethasone in order to identify molecules that can reverse the anemic phenotype., (Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with β-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients., (© 2015 by The American Society of Hematology.)

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein-protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs., (© 2014 Lin, Ear, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Diamond-Blackfan anemia (DBA) is a rare congenital red cell aplasia that classically presents during early infancy in DBA patients. Approximately, 25% of patients carry a mutation in the ribosomal protein (RP) S19 gene; mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 have been reported. How ribosome protein deficiency causes defects specifically to red blood cells in DBA has not been well elucidated. To genetically model the predominant ribosome defect in DBA, we generated an rps19 null mutant through the use of TALEN-mediated gene targeting in zebrafish. Molecular characterization of this mutant line demonstrated that rps19 deficiency reproduced the erythroid defects of DBA, including a lack of mature red blood cells and p53 activation. Notably, we found that rps19 mutants' production of globin proteins was significantly inhibited; however, globin transcript level was either increased or unaffected in rps19 mutant embryos. This dissociation of RNA/protein levels of globin genes was confirmed in another zebrafish DBA model with defects in rpl11. Using transgenic zebrafish with specific expression of mCherry in erythroid cells, we showed that protein production in erythroid cells was decreased when either rps19 or rpl11 was mutated. L-Leucine treatment alleviated the defects of protein production in erythroid cells and partially rescued the anemic phenotype in both rps19 and rpl11 mutants. Analysis of this model suggests that the decreased protein production in erythroid cells likely contributes to the blood-specific phenotype of DBA. Furthermore, the newly generated rps19 zebrafish mutant should serve as a useful animal model to study DBA. Our in vivo findings may provide clues for the future therapy strategy for DBA.

The zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. In this Communication, the implementation of a roboticized high-throughput screening (HTS) platform with automated image analysis is demonstrated to assess the impact of dissolvable oxide nanoparticles on embryo hatching. It is further demonstrated that this hatching interference is mechanistically linked to an effect on the metalloprotease, ZHE 1, which is responsible for degradation of the chorionic membrane. The data indicate that 4 of 24 metal oxide nanoparticles (CuO, ZnO, Cr2 O3 , and NiO) could interfere with embryo hatching by a chelator-sensitive mechanism that involves ligation of critical histidines in the ZHE1 center by the shed metal ions. A recombinant ZHE1 enzymatic assay is established to demonstrate that the dialysates from the same materials responsible for hatching interference also inhibit ZHE1 activity in a dose-dependent fashion. A peptide-based BLAST search identifies several additional aquatic species that express enzymes with homologous histidine-based catalytic centers, suggesting that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of oxide nanoparticles that pose a hazard to aquatic species., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The synaptic scaffolding proteins CASK and Caskin1 are part of the fibrous mesh of proteins that organize the active zones of neural synapses. CASK binds to a region of Caskin1 called the CASK interaction domain (CID). Adjacent to the CID, Caskin1 contains two tandem sterile α motif (SAM) domains. Many SAM domains form polymers so they are good candidates for forming the fibrous structures seen in the active zone. We show here that the SAM domains of Caskin1 form a new type of SAM helical polymer. The Caskin1 polymer interface exhibits a remarkable segregation of charged residues, resulting in a high sensitivity to ionic strength in vitro. The Caskin1 polymers can be decorated with CASK proteins, illustrating how these proteins may work together to organize the cytomatrix in active zones., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

GIV (Gα-interacting vesicle-associated protein; also known as Girdin) enhances Akt activation downstream of multiple growth factor- and G protein (heterotrimeric guanosine 5'-triphosphate-binding protein)-coupled receptors to trigger cell migration and cancer invasion. We demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at tyrosine-1764 and tyrosine-1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the amino- and carboxyl-terminal Src homology 2 domains of p85α, a regulatory subunit of PI3K; stabilized receptor association with PI3K; and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85α increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIV-PI3K interaction a potential therapeutic target within the PI3K-Akt pathway.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a conserved multi-domain scaffolding protein involved in brain development, synapse formation, and establishment of cell polarity. To accomplish these diverse functions, CASK participates in numerous protein-protein interactions. In particular, CASK forms competing CASK/Mint1/Velis and CASK/Caskin1/Velis tripartite complexes that physically associate with the cytoplasmic tail of neurexin, a transmembrane protein enriched at presynaptic sites. This study shows that a short linear EEIWVLRK peptide motif from Caskin1 is necessary and sufficient for binding CASK. We also identified the conserved binding site for the peptide on the CASK calmodulin kinase domain. A related EPIWVMRQ peptide from Mint1 was also discovered to be sufficient for binding. Searching all human proteins for the Mint1/Caskin1 consensus peptide ExIWVxR revealed that T-cell lymphoma invasion and metastasis 1 (TIAM1) contains a conserved EEVIWVRRE peptide that was also found to be sufficient for CASK binding in vitro. TIAM1 is well known for its role in tumor metastasis, but it also possesses overlapping cellular and neurological functions with CASK, suggesting a previously unknown cooperation between the two proteins. This new peptide interaction motif also explains how Caskin1 and Mint1 form competing complexes and suggests a new role for the cellular hub protein CASK., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Autophagy is the major catabolic process responsible for the removal of aggregated proteins and damaged organelles. Autophagy is regulated by both G proteins and growth factors, but the underlying mechanism of how they are coordinated during initiation and reversal of autophagy is unknown. Using protein-protein interaction assays, G protein enzymology, and morphological analysis, we demonstrate here that Gα-interacting, vesicle-associated protein (GIV, a. k. a. Girdin), a nonreceptor guanine nucleotide exchange factor for Gα(i3), plays a key role in regulating autophagy and that dynamic interplay between Gα(i3), activator of G-protein signaling 3 (AGS3, its guanine nucleotide dissociation inhibitor), and GIV determines whether autophagy is promoted or inhibited. We found that AGS3 directly binds light chain 3 (LC3), recruits Gα(i3) to LC3-positive membranes upon starvation, and promotes autophagy by inhibiting the G protein. Upon growth factor stimulation, GIV disrupts the Gα(i3)-AGS3 complex, releases Gα(i3) from LC3-positive membranes, enhances anti-autophagic signaling pathways, and inhibits autophagy by activating the G protein. These results provide mechanistic insights into how reversible modulation of Gα(i3) activity by AGS3 and GIV maintains the delicate equilibrium between promotion and inhibition of autophagy.

Metastasis accounts for the majority of cancer-related deaths. Accurate prediction of metastatic potential of tumors has been elusive, and the search for clinically useful markers continues. We previously reported that GIV/Girdin triggers tumor cell migration by virtue of a C-terminal guanine-nucleotide exchange factor motif that activates Gαi. Here we identify GIV as a metastasis-related protein whose full-length transcript (GIV-fl) is expressed exclusively in highly invasive colon, breast, and pancreatic carcinoma cells and not in their poorly invasive counterparts. A prospective, exploratory biomarker study conducted on a cohort of 56 patients with stage II colorectal cancer revealed a significant correlation between GIV-fl expression in tumor epithelium and shortened metastasis-free survival. Survival rate for patients with GIV-fl-positive tumors is significantly reduced compared with the patients with GIV-fl-negative tumors [P<0.0001; hazard ratio=0.076; CI=0.052-0.30 (95%)]. At the 5-yr mark, survival is 100% in the GIV-fl-negative group and 62 ± 9% (mean±SE; P=6×10(-5)) in the GIV-fl-positive group. Furthermore, GIV-fl expression predicts a risk of mortality independent of the microsatellite stability status, a well-established prognosticator of colorectal cancers. We conclude that GIV-fl is a novel metastasis-related protein and an independent adverse prognosticator that may serve as a useful adjunct to traditional staging strategies in colorectal carcinoma.

Cells respond to growth factors by either migrating or proliferating, but not both at the same time, a phenomenon termed migration-proliferation dichotomy. The underlying mechanism of this phenomenon has remained unknown. We demonstrate here that Galpha(i) protein and GIV, its nonreceptor guanine nucleotide exchange factor (GEF), program EGF receptor (EGFR) signaling and orchestrate this dichotomy. GIV directly interacts with EGFR, and when its GEF function is intact, a Galpha(i)-GIV-EGFR signaling complex assembles, EGFR autophosphorylation is enhanced, and the receptor's association with the plasma membrane (PM) is prolonged. Accordingly, PM-based motogenic signals (PI3-kinase-Akt and PLCgamma1) are amplified, and cell migration is triggered. In cells expressing a GEF-deficient mutant, the Galphai-GIV-EGFR signaling complex is not assembled, EGFR autophosphorylation is reduced, the receptor's association with endosomes is prolonged, mitogenic signals (ERK 1/2, Src, and STAT5) are amplified, and cell proliferation is triggered. In rapidly growing, poorly motile breast and colon cancer cells and in noninvasive colorectal carcinomas in situ in which EGFR signaling favors mitosis over motility, a GEF-deficient splice variant of GIV was identified. In slow growing, highly motile cancer cells and late invasive carcinomas, GIV is highly expressed and has an intact GEF motif. Thus, inclusion or exclusion of GIV's GEF motif, which activates Galphai, modulates EGFR signaling, generates migration-proliferation dichotomy, and most likely influences cancer progression.

Although several non-receptor activators of heterotrimeric G proteins have been identified, the structural features of G proteins that determine their interaction with such activators and the subsequent biological effects are poorly understood. Here we investigated the structural determinants in G alpha(i3) necessary for its regulation by GIV/girdin, a guanine-nucleotide exchange factor (GEF) that activates G alpha(i) subunits. Using G protein activity and in vitro pulldown assays we demonstrate that G alpha(i3) is a better substrate for GIV than the highly homologous G alpha(o). We identified Trp-258 in the G alpha(i) subunit as a novel structural determinant for GIV binding by comparing GIV binding to G alpha(i3)/G alpha(o) chimeras. Mutation of Trp-258 to the corresponding Phe in G alpha(o) decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other G alpha-binding partners, i.e. G betagamma, AGS3 (a guanine nucleotide dissociation inhibitor), GAIP/RGS19 (a GTPase-activating protein), and LPAR1 (a G protein-coupled receptor). Activation of G alpha(i3) by GIV was also dramatically reduced when Trp-258 was replaced with Tyr, Leu, Ser, His, Asp, or Ala, highlighting that Trp is required for maximal activation. Moreover, when mutant G alpha(i3) W258F was expressed in HeLa cells they failed to undergo cell migration and to enhance Akt signaling after growth factor or G protein-coupled receptor stimulation. Thus activation of G alpha(i3) by GIV is essential for biological functions associated with G alpha(i3) activation. In conclusion, we have discovered a novel structural determinant on G alpha(i) that plays a key role in defining the selectivity and efficiency of the GEF activity of GIV on G alpha(i) and that represents an attractive target site for designing small molecules to disrupt the G alpha(i)-GIV interface for therapeutic purposes.