Role Of Notch Signaling Research Articles

Role for notch signaling in salivary acinar cell growth and differentiation

The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of gamma-secretase, which is required for Notch cleavage and activation, blocked vimentin and cystatin S expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downstream signal Hes-1 expression, and Hes-1 expression was inhibited by gamma-secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes-1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation.

Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells

AbstractNotch signaling is involved in tumorigenesis, but its role in B–chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor–kappa B (NF-κB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the γ-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-κB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

The role of NOTCH1 signaling in T-ALL

The identification of activating mutations in NOTCH1 in over 50% of T-cell acute lymphoblastic leukemias (T-ALL) has generated major interest in the elucidation of the mechanisms of transformation downstream of oncogenic NOTCH and in the targeting of the NOTCH signaling pathway in this disease. Small molecule gamma-secretase inhibitors (GSIs) block NOTCH1 signaling in T-ALL lymphoblasts, yet the clinical development of GSIs has been held back by the development of gastrointestinal toxicity and their weak antileukemic effects against human T-ALL. However, new therapeutic strategies aiming to optimize the use of anti-NOTCH1 therapies for T-ALL, including combination therapies with molecularly targeted drugs and glucocorticoids, have started to emerge as a result of improved understanding of the molecular mechanisms that mediate the effects of GSIs in leukemic cells and the intestinal epithelium. This review focuses on the molecular basis of NOTCH1-induced transformation, the mechanisms of action of oncogenic NOTCH1 and clinical significance of NOTCH1 mutations in T-ALL.

Notch signaling confers antigen-presenting cell functions on mast cells

Notch signaling is involved in cell fate determination along with the development of the immune system. However, very little is known about the role for Notch signaling in mast cells. We investigated the role of Notch signaling in mast cell functions. After mouse bone marrow-derived mast cells (BMMCs) or peritoneal mast cells (PMCs) were cocultured with mouse Notch ligand-expressing chinese hamster ovary cells for 5 days, we examined the mast cell surface expressions of MHC-II molecules and OX40 ligand (OX40L), Fc epsilon RI-mediated cytokine production, and the effects of the mast cells on proliferation and differentiation of naive CD4(+) T cells in vitro. We showed that BMMCs and PMCs constitutively expressed Notch1 and Notch2 proteins on the cell surface. We also found that Delta-like 1 (Dll1)/Notch signaling induced the expression of MHC-II and upregulated the expression level of OX40L on the surface of the mast cells. Dll1/Notch signaling augmented Fc epsilon RI-mediated IL-4, IL-6, IL-13, and TNF production by BMMCs. Dll1-stimulated MHC-II(+)OX40L(high) BMMCs promoted proliferation of naive CD4(+) T cells and their differentiation into T(H)2 cells producing IL-4, IL-5, IL-10, and IL-13. Dll1/Notch signaling confers the functions as an antigen-presenting cell on mast cells, which preferentially induce the differentiation of T(H)2.

Differential gene expression in a coculture model of angiogenesis reveals modulation of select pathways and a role for Notch signaling

Communication between endothelial and mural cells (smooth muscle cells, pericytes, and fibroblasts) can dictate blood vessel size and shape during angiogenesis, and control the functional aspects of mature blood vessels, by determining things such as contractile properties. The ability of these different cell types to regulate each other's activities led us to ask how their interactions directly modulate gene expression. To address this, we utilized a three-dimensional model of angiogenesis and screened for genes whose expression was altered under coculture conditions. Using a BeadChip array, we identified 323 genes that were uniquely regulated when endothelial cells and mural cells (fibroblasts) were cultured together. Data mining tools revealed that differential expression of genes from the integrin, blood coagulation, and angiogenesis pathways were overrepresented in coculture conditions. Scans of the promoters of these differentially modulated genes identified a multitude of conserved C promoter binding factor (CBF)1/CSL elements, implicating Notch signaling in their regulation. Accordingly, inhibition of the Notch pathway with gamma-secretase inhibitor DAPT or NOTCH3-specific small interfering RNA blocked the coculture-induced regulation of several of these genes in fibroblasts. These data show that coculturing of endothelial cells and fibroblasts causes profound changes in gene expression and suggest that Notch signaling is a critical mediator of the resultant transcription.

Notch signaling in pediatric malignancies

Notch signaling plays crucial roles in many developmental pathways, with Notch mutations linked to several developmental disorders. Because many pediatric malignancies arise from dysregulated development, roles for Notch signaling in these cancers are to be expected. Evidence to support this is now emerging as the Notch pathway is being explored in more pediatric cancers. Not surprisingly, Notch appears to play diverse roles in different malignancies, effecting differentiation, metastasis, cancer "stem cells," and angiogenesis. As examples, although activating mutations of Notch1 are found in the majority of T-cell acute lymphoblastic leukemia (ALL) cases, Notch/HES1 signaling appears to play a tumor suppressor role in precursor B-cell ALL; although Notch/HES1 signaling appears to contribute to osteosarcoma metastasis, Notch signaling also promotes medulloblastoma "stem cell" survival and contributes to angiogenesis in neuroblastoma. Further understanding of the roles of Notch signaling in specific pediatric cancers will provide a rationale for Notch-based therapeutic strategies.

Abstract 1680: Notch Signaling Directly Targets Msx2: Possible Role of Notch Signaling in Osteogenic Conversion of Vascular Smooth Muscle Cells and Vascular Calcification

Vascular calcification is a prominent feature of atherosclerosis and closely correlated with cardiovascular morbidity and mortality. In this study, we hypothesize that Notch signaling plays an important role in osteogenic conversion of smooth muscle cells (SMCs) and vascular calcification. <Methods and Results> Either Notch ligand-expressing cells or overexpression of Notch intracellular domains (NICDs) induced expression of Msx2, a key regulator of osteogenic conversion, in human aortic SMCs (HASMCs). In addition, overexpression of Notch1 intracellular domain (N1-ICD) markedly upregulated alkaline phosphatase (ALP) activity and matrix mineralization of HASMCs. A knockdown experiment with a small interfering RNA confirmed that Msx2, but not Runx2/Cbfa1, another key osteogenic transcription factor, is responsible for Notch1-induced osteogenic conversion of HASMCs. Furthermore, this Notch1-Msx2 pathway was independent of bone morphogenetic protein-2 (BMP-2), an osteogenic morphogen upstream of Msx2. The transcriptional activity of the Msx2 promoter was significantly enhanced by Notch ligands stimulation, whereas it was abrogated by a specific Notch signaling inhibitor. The RBP-Jk binding element within the Msx2 promoter was critical to Notch1-induced Msx2 gene expression, and correspondingly, neither N1-ICD overexpression nor Notch ligands stimulation increase the Msx2 expression or transcriptional activity of the Msx2 promoter, respectively, in RBP-Jk-deficient fibroblasts. Immunohistochemistry of human artery specimens revealed colocalization of Notch1 and Msx2 within atherosclerotic plaques, indicating a role of Notch1-Msx2 pathway in vascular calcification in vivo. These results suggest that Notch signaling directly targets Msx2, thus accelerating osteogenic conversion of HASMCs and, as a result, a formation of vascular calcification.

Ero1L, a thiol oxidase, is required for Notch signaling through cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster

Notch-mediated cell–cell communication regulates numerous developmental processes and cell fate decisions. Through a mosaic genetic screen in Drosophila melanogaster, we identified a role in Notch signaling for a conserved thiol oxidase, endoplasmic reticulum (ER) oxidoreductin 1–like (Ero1L). Although Ero1L is reported to play a widespread role in protein folding in yeast, in flies Ero1L mutant clones show specific defects in lateral inhibition and inductive signaling, two characteristic processes regulated by Notch signaling. Ero1L mutant cells accumulate high levels of Notch protein in the ER and induce the unfolded protein response, suggesting that Notch is misfolded and fails to be exported from the ER. Biochemical assays demonstrate that Ero1L is required for formation of disulfide bonds of three Lin12-Notch repeats (LNRs) present in the extracellular domain of Notch. These LNRs are unique to the Notch family of proteins. Therefore, we have uncovered an unexpected requirement for Ero1L in the maturation of the Notch receptor.

A Role for Notch Signaling in Stromal Survival and Differentiation during Prostate Development

Notch1 signaling is involved in epithelial growth and differentiation of prostate epithelia, and we have examined the role that notch signaling plays in the stroma of the developing prostate. We initially observed expression of delta-like 1 (Dlk1) and Notch2 in gene profiling studies of prostatic mesenchyme, and anticipated that they might be expressed in a key subset of inductive mesenchyme. Using quantitative RT-PCR, Northern blotting, and whole mount in situ hybridization, we confirmed that both Dlk1 and Notch2 mRNAs showed a restricted expression pattern within subsets of the stroma during prostate development. Localization of Dlk1 and Notch2 proteins mirrored the transcript expression, and showed both distinct and overlapping expression patterns within the stroma. Dlk1 and Notch2 were coexpressed in condensed inductive mesenchyme of the ventral mesenchymal pad (VMP), and were partially colocalized in the smooth muscle (SM) layer of the urethral stroma. In addition, Dlk1 was not expressed in SM adjacent to the VMP in female urethra. The function of notch signaling was examined using organ cultures of prostate rudiments and a small molecule inhibitor of notch receptor activity. Inhibition of notch signaling led to a loss of stromal tissue in both prostate and female VMP cultures, suggesting that this pathway was required for stromal survival. Inhibition of notch signaling also led to changes in both epithelial and stromal differentiation, which was evident in altered distributions of SM alpha-actin and p63 in prostates grown in vitro. The effects of notch signaling upon the stroma were only evident in the presence of testosterone, in contrast to effects upon epithelial differentiation.

A deficiency of lunatic fringe is associated with cystic dilation of the rete testis

Lunatic fringe belongs to a family of beta1-3 N-acetyltransferases that modulate the affinity of the Notch receptors for their ligands through the elongation of O-fucose moieties on their extracellular domain. A role for Notch signaling in vertebrate fertility has been predicted by the intricate expression of the Notch receptors and their ligands in the oocyte and granulosa cells of the ovary and the spermatozoa and Sertoli cells of the testis. It has been demonstrated that disruption of Notch signaling by inactivation of lunatic fringe led to infertility associated with pleiotropic defects in follicle development and meiotic maturation of oocytes. Lunatic fringe null males were found to be subfertile. Here, we report that gene expression data demonstrate that fringe and Notch signaling genes are expressed in the developing testis and the intratesticular ductal tract, predicting roles for this pathway during embryonic gonadogenesis and spermatogenesis. Spermatogenesis was not impaired in the majority of the lunatic fringe null males; however, spermatozoa were unilaterally absent in the epididymis of many mice. Histological and immunohistochemical analysis of these testes revealed the development of unilateral cystic dilation of the rete testis. Tracer dye experiments confirm a block in the connection between the rete testis and the efferent ducts. Further, the dye studies demonstrated that many lunatic fringe mutant males had partial blocks of the connection between the rete testis and the efferent ducts bilaterally.

The role of Notch signaling in human cervical cancer: implications for solid tumors

The detection of intracellular forms of Notch1 in human cervical cancers more than a decade ago prompted an investigation into the possible role of this pathway in driving these cancers. These tumors are consistently characterized by features of deregulated ligand-dependent signaling. Although Notch signaling complements the function of papillomavirus oncogenes in transformation assays of human keratinocytes, there are dose-dependent effects, which inhibit growth of established cervical cancer cell lines. Two pro-oncogenic effector mechanisms that have been suggested to operate in this context by Notch signaling are the activation of PI3K/Akt pathway and the upregulation of c-Myc. Collectively, there is a complex interplay between Notch signaling and papillomaviruses in the context of cervical carcinogenesis. Better animal model systems and identification of human cervical cancer stem cells should help clarify the possible stage specific and pleiotropic effects and regulation of Notch signaling.

Soluble Forms of the Notch Ligands Delta1 and Jagged1 Promote in Vivo Tumorigenicity in NIH3T3 Fibroblasts with Distinct Phenotypes

We previously found that soluble forms of the Notch ligands Jagged1 and Delta1 induced fibroblast growth factor receptor-dependent cell transformation in NIH3T3 fibroblasts. However, the phenotypes of these lines differed, indicating distinct functional differences among these Notch ligands. In the present study, we used allografts to test the hypothesis that NIH3T3 fibroblasts that express soluble forms of Delta1 and Jagged1 accelerate tumorigenicity in vivo. With the exception of the full-length Jagged1 transfectant, all other cell lines, including the control, generated tumors when injected subcutaneously in athymic mice. Suppression of Notch signaling by the soluble ligands significantly increased tumor onset and growth, whereas full-length Jagged1 completely suppressed tumor development. In addition, there were striking differences in tumor pathology with respect to growth kinetics, vascularization, collagen content, size and number of necrotic foci, and invasiveness into the underlying tissue. Further, the production of angiogenic factors, including vascular endothelial growth factor, also differed among the tumor types. Lastly, both Jagged1- and Delta1-derived tumors contained phenotypically distinct populations of lipid-filled cells that corresponded with increased expression of adipocyte markers. The divergence of tumor phenotype may be attributed to ligand-specific alterations in Notch receptor responses in exogenous and endogenous cell populations within the allographs. Our findings demonstrate distinct functional properties for these Notch ligands in the promotion of tumorigenicity in vivo.

Notch inhibition promotes human embryonic stem cell-derived cardiac mesoderm differentiation.

The roles of Notch signaling in cardiac differentiation from murine embryonic stem cells have been well documented. We investigated whether Notch signaling plays a similar role in human embryonic stem cells (hESCs). Although, as previously reported, blocking Notch signaling via the addition of gamma-secretase inhibitor (GSI) alone failed to affect hESC differentiation, we found that GSI plus reduced-volume culture medium (GSI/RVCM) accelerated mesodermal differentiation. GSI/RVCM conditions simultaneously suppressed commitment toward neuroectodermal lineages. Furthermore, sustained inhibition of Notch signaling further enhanced differentiation into cardiac mesoderm. Spontaneous beating activity was typically observed from 12 days after initiation of GSI treatment in RVCM. Moreover, hESC-derived cardiomyocytes expressed connexin 43 and possessed spontaneous calcium oscillations and cardiomyocyte beats coupled to neonatal rat cardiomyocytes when cocultured. These findings strongly suggest a distinct role for Notch signaling in the induction and specification of hESC-derived cardiac mesoderm in vitro. Disclosure of potential conflicts of interest is found at the end of this article.

MultipleNotchsignaling events controlDrosophilaCNS midline neurogenesis, gliogenesis and neuronal identity

The study of how transcriptional control and cell signaling influence neurons and glia to acquire their differentiated properties is fundamental to understanding CNS development and function. The Drosophila CNS midline cells are an excellent system for studying these issues because they consist of a small population of diverse cells with well-defined gene expression profiles. In this paper, the origins and differentiation of midline neurons and glia were analyzed. Midline precursor (MP) cells each divide once giving rise to two neurons; here, we use a combination of single-cell gene expression mapping and time-lapse imaging to identify individual MPs, their locations, movements and stereotyped patterns of division. The role of Notch signaling was investigated by analyzing 37 midline-expressed genes in Notch pathway mutant and misexpression embryos. Notch signaling had opposing functions: it inhibited neurogenesis in MP1,3,4 and promoted neurogenesis in MP5,6. Notch signaling also promoted midline glial and median neuroblast cell fate. This latter result suggests that the median neuroblast resembles brain neuroblasts that require Notch signaling, rather than nerve cord neuroblasts, the formation of which is inhibited by Notch signaling. Asymmetric MP daughter cell fates also depend on Notch signaling. One member of each pair of MP3-6 daughter cells was responsive to Notch signaling. By contrast, the other daughter cell asymmetrically acquired Numb, which inhibited Notch signaling, leading to a different fate choice. In summary, this paper describes the formation and division of MPs and multiple roles for Notch signaling in midline cell development, providing a foundation for comprehensive molecular analyses.

OSM-11 Facilitates LIN-12 Notch Signaling during Caenorhabditis elegans Vulval Development

Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.

Notch Signaling Is Involved in Expression of Thyrocyte Differentiation Markers and Is Down-Regulated in Thyroid Tumors

Notch genes encode receptors for a signaling pathway that regulates cell growth and differentiation in various contexts, but the role of Notch signaling in thyroid follicular cells has never been fully published. The objective of the study was to characterize the expression of Notch pathway components in thyroid follicular cells and Notch signaling activities in normal and transformed thyrocytes. DESIGN/SETTING AND PATIENTS: Expression of Notch pathway components and key markers of thyrocyte differentiation was analyzed in murine and human thyroid tissues (normal and tumoral) by quantitative RT-PCR and immunohistochemistry. The effects of Notch overexpression in human thyroid cancer cells and FTRL-5 cells were explored with analysis of gene expression, proliferation assays, and experiments involving transfection of a luciferase reporter construct containing human NIS promoter regions. Notch receptors are expressed during the development of murine thyrocytes, and their expression levels parallel those of thyroid differentiation markers. Notch signaling characterized also normal adult thyrocytes and is regulated by TSH. Notch pathway components are variably expressed in human normal thyroid tissue and thyroid tumors, but expression levels are clearly reduced in undifferentiated tumors. Overexpression of Notch-1 in thyroid cancer cells restores differentiation, reduces cell growth rates, and stimulates NIS expression via a direct action on the NIS promoter. Notch signaling is involved in the determination of thyroid cell fate and is a direct regulator of thyroid-specific gene expression. Its deregulation may contribute to the loss of differentiation associated with thyroid tumorigenesis.

Notch‐regulated oligodendrocyte specification from radial glia in the spinal cord of zebrafish embryos

During vertebrate neural development, many dividing neuroepithelial precursors adopt features of radial glia, which are now known to also serve as neural precursors. In mammals, most radial glia do not persist past early postnatal stages, whereas zebrafish maintain large numbers of radial glia into adulthood. The mechanisms that maintain and specify radial glia for different fates are still poorly understood. We investigated formation of radial glia in the spinal cord of zebrafish and the role of Notch signaling in their maintenance and specification. We found that spinal cord precursors begin to express gfap+, a marker of radial glia, during neurogenesis and that gfap cells give rise to both neurons and oligodendrocytes. We also determined that Notch signaling is continuously required during embryogenesis to maintain radial glia, limit motor neuron formation and permit oligodendrocyte development, but that radial glia seem to be refractory to changes in Notch activity in postembryonic animals.

Notch Presents a Challenge

In patients with asthma, inflammation and airway hyperresponsiveness (AHR) are associated with the accumulation of CD8 + T cells in the lungs. Mouse models of asthma involve administering an allergen to the airway (sensitization), followed by a second exposure to allergen (challenge), which triggers AHR. Okamoto et al . previously showed that AHR was less severe in CD8 –/– mice than in wild-type mice and could be restored by the transfer of CD8 + effector (T EFF ), but not CD8 + central memory (T CM ), T cells to the CD8 –/– mice. In the current study, microarray analyses showed that Notch1 mRNA was more abundant in T EFF than in T CM cells. Notch receptors determine cell fate in developmental processes, including during T cell lineage commitment. Activation of Notch receptors by their ligands, members of the Delta-like and Jagged families, results in γ-secretase-dependent cleavage of the Notch intracellular domain (NICD), which translocates to the nucleus to activate target gene transcription. Stimulation of the T cell receptors of T EFF cells in vitro increased the abundance of NICD compared with that in untreated cells, as determined by Western blotting. When T EFF cells were treated in vitro with a γ-secretase inhibitor (GSI) before transfer to allergen-challenged CD8 –/– mice, AHR was less severe than that in mice that received vehicle-treated T EFF cells. Finally, when allergen-sensitized wild-type mice were treated with an antibody against the Notch ligand Delta1 before allergen challenge, their AHR was less severe than that of control antibody-treated mice. Together, these data indicate a role for Notch signaling in the development of CD8 + T cell-mediated AHR and suggest Delta1 as a potential therapeutic target in the treatment of allergen-induced AHR. M. Okamoto, K. Takeda, A. Joetham, H. Ohnishi, H. Matsuda, C. H. Swasey, B. J. Swanson, K. Yasutomo, A. Dakhama, E. W. Gelfand, Essential role of Notch signaling in effector memory CD8 + T cell-mediated airway hyperresponsiveness and inflammation. J . Exp . Med . 205 , 1087-1097 (2008). [Abstract] [Full Text]

Notch Signaling Activation in Human Embryonic Stem Cells Is Required for Embryonic, but Not Trophoblastic, Lineage Commitment

The Notch signaling pathway plays important roles in cell-fate determination during embryonic development and adult life. In this study, we focus on the role of Notch signaling in governing cell-fate choices in human embryonic stem cells (hESCs). Using genetic and pharmacological approaches, we achieved both blockade and conditional activation of Notch signaling in several hESC lines. We report here that activation of Notch signaling is required for undifferentiated hESCs to form the progeny of all three embryonic germ layers, but not trophoblast cells. In addition, transient Notch signaling pathway activation enhanced generation of hematopoietic cells from committed hESCs. These new insights into the roles of Notch in hESC-fate determination may help to efficiently direct hESC differentiation into therapeutically relevant cell types.

Prevalence of Intrinsic Disorder in the Intracellular Region of Human Single-Pass Type I Proteins: The Case of the Notch Ligand Delta-4

Intrinsic disorder (ID) is a widespread phenomenon found especially in signaling and regulation-related eukaryotic proteins. The functional importance of flexible disordered regions often resides in their ability to allow proteins to bind different partners. The incidence and location of intrinsic disorder in 369 human single-pass transmembrane receptors with the type I topology was assessed based on both disorder predictions and amino acid physico-chemical properties. We provide evidence that ID concentrates in the receptors' cytoplasmic region. As a benchmark for this analysis, we present a structural study on the previously uncharacterized intracellular region of human Delta-4 (DLL4_IC), a single-pass transmembrane protein and a ligand of Notch receptors. DLL4_IC is required for receptor/ligand endocytosis; it undergoes regulated intramembrane proteolysis, and mediates protein-protein interactions through its C-terminal PDZ binding motif. Using a recombinant purified protein, we demonstrate using various biophysical methods that DLL4_IC is mainly disordered in solution but can form interconvertible local secondary structures in response to variations in the physico-chemical milieu. Most of these conformational changes occur in the highly conserved C-terminal segment that includes the PDZ-binding motif. On the basis of our results, we propose that global disorder, in concert with local preorganization, may play a role in Notch signaling mediated by Delta-4.