scholarly journals Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling

2019 ◽  
Vol 12 (606) ◽  
pp. eaay2369 ◽  
Author(s):  
Sanchez M. Jarrett ◽  
Tom C. M. Seegar ◽  
Mark Andrews ◽  
Guillaume Adelmant ◽  
Jarrod A. Marto ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Negative Feedback ◽  
Transcriptional Activation ◽  
Cultured Cells ◽  
Feedback Inhibition ◽  
Lymphoblastic Leukemia ◽  
Ankyrin Repeat ◽  
Structural Basis ◽  
Intracellular Domain ◽  
Notch Intracellular Domain

Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.

scholarly journals Molecular basis for feedback inhibition of Notch signaling by the Notch regulated ankyrin repeat protein NRARP

2019 ◽  
Author(s):  
Sanchez M. Jarrett ◽  
Tom C. M. Seegar ◽  
Mark Andrews ◽  
Guillaume Adelmant ◽  
Jarrod A. Marto ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Transcriptional Activation ◽  
Molecular Basis ◽  
Feedback Inhibition ◽  
Ankyrin Repeat ◽  
Structural Basis ◽  
Repeat Protein ◽  
Ankyrin Repeat Protein ◽  
Notch Intracellular Domain ◽  
Binding Interface

AbstractThe Notch regulated ankyrin repeat protein (NRARP) is a negative feedback regulator of Notch signaling in higher organisms. The molecular basis for NRARP function, however, has remained elusive. Mass spectrometry-based proteomic studies show that human NRARP associates with the core Notch transcriptional activation complex (NTC), containing the RBPJ transcription factor, the Notch intracellular domain (NICD), and a Mastermind-like co-activator. Binding of NRARP is direct, requires both RBPJ and NICD, and is independent of Mastermind-like proteins or DNA. The X-ray structure of an NRARP/RBPJ/NOTCH1/DNA complex, determined to 3.75 Å resolution, reveals a non-canonical mode of binding in which NRARP extends the NOTCH1 ankyrin-repeat stack by three additional repeats. Mutations of NRARP residues at the binding interface disrupt entry into complexes and suppress feedback inhibition in signaling assays. These studies establish the structural basis for NTC engagement by NRARP and provide insight into its mechanism of feedback inhibition of Notch signaling.

scholarly journals Dimerization of the Notch Intracellular Domain Results in Distinct Signaling Activity

2020 ◽  
Author(s):  
Jacob Jeffery Crow
Keyword(s):  
Notch Signaling ◽  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Regulatory Mechanism ◽  
Notch Signaling Pathway ◽  
Core Component ◽  
High Tunability ◽  
Notch Intracellular Domain ◽  
Signaling Field

The Notch signaling pathway is a core component of multicellularity; enabling cells to directly communicate with both their neighbors and the surrounding microenvironment. These signals are translated directly through the Notch proteins, where a fragment of Notch transitions into the nucleus to act as a co-transcription factor, setting into motion a host of physiological responses. Commonly involved in pathways that define a cell’s identity and fate decisions, what appears to be a simplistic pathway instead exists in a state of high-tunability and strict control. Missteps in this pathway are generally embryonically lethal or lead to a suite of congenital disorders and cancers. Therefore, it’s pertinent to understand the mechanisms of Notch that provide its flexibility and pleiotropic outcomes. One such property is its ability to homodimerize on DNA while within its transcriptional activation complex, resulting in an enhanced transcriptional signal of a select pool of Notch target genes. This dissertation reviews the general mechanics behind Notch signaling, discusses how the field of Notch dimerization came to be and where it stands currently, and finally, details my contributions to the understanding of this regulatory mechanism. Despite Notch’s ubiquitous function in metazoan life, there are still many mysteries behind this signaling pathway. The work detailed here describes my time spent as a basic science researcher, where my findings contribute a couple of puzzle pieces to the expansive Notch signaling field.

scholarly journals Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites

2020 ◽  
Author(s):  
Jacob J. Crow ◽  
Allan R. Albig
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Regulatory Mechanism ◽  
Family Members ◽  
Regulatory Function ◽  
Specific Nature ◽  
Intracellular Domain ◽  
Notch Intracellular Domain ◽  
Highly Sensitive ◽  
Cellular Identity

ABSTRACTNotch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch’s ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that each family member is capable of dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members’ SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.

The Notch signalling pathway and breast cancer: The importance of balance and cellular self-control.

2019 ◽  
Vol 14 ◽  
Author(s):  
Germán Saucedo-Correa ◽  
Alejandro Bravo-Patiño ◽  
Rosa Elvira Núñez-Anita ◽  
Javier Oviedo-Boyso ◽  
Juan José Valdez-Alarcón ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Signaling Pathway ◽  
Cross Talk ◽  
Transcriptional Activation ◽  
Notch Pathway ◽  
Notch Signaling Pathway ◽  
Self Control ◽  
Design Strategies ◽  
Notch Intracellular Domain ◽  
The Cross

Notch is a cell-signaling pathway that is highly conserved in all metazoans and is responsible for cell differentiation and cross-talk communication with other signaling pathways such as WNT and Hh. In most cancers, the Notch signaling pathway is altered, causing atypical activity of vital processes such as cell cycle, differentiation and apoptosis, leading the cell to a carcinogenic state. Currently, the Notch signaling pathway has taken a special interest to design strategies in order to regulate the activity of this pathway since it is known that in the cancer molecular micro-environment the Notch pathway is over-expressed or presents an aberrant function, which, in consequence, corrupts the cross-talk communication with WNT and Hh pathways. Most of the existing strategies are focused on the systematic and whole inhibition of Notch pathway at the membrane level by the use of γ-secretases inhibitors. There are few strategies that act at the nuclear level inhibiting the activity of the transcriptional activation complex composed by the Notch intracellular domain, the transcriptional factor CSL and the Mastermind co-activator. In this review, by the fact that there are not any strategy focused to revert the over expression effect caused by the Notch pathway constitutive activity, we propose that the efforts to develop new strategies against cancer should be focused to understand the complexity of the cross-talk communication between Notch, WNT and Hh pathways to neutralize the gene aberrant activity characteristic of cancer cells which are responsible for those corrupted cross-talk communication.

scholarly journals NUMB enhances Notch signaling by repressing ubiquitination of NOTCH1 intracellular domain

2019 ◽  
Vol 12 (5) ◽  
pp. 345-358 ◽  
Author(s):  
Zhiyuan Luo ◽  
Lili Mu ◽  
Yue Zheng ◽  
Wenchen Shen ◽  
Jiali Li ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Posttranslational Modifications ◽  
Transcriptional Activation ◽  
Neural Progenitor Cells ◽  
Nuclear Translocation ◽  
Notch Receptor ◽  
Deubiquitinating Enzyme ◽  
Intracellular Domain ◽  
Ubiquitin Proteasome ◽  
Notch1 Receptor

Abstract The release and nuclear translocation of the intracellular domain of Notch receptor (NICD) is the prerequisite for Notch signaling-mediated transcriptional activation. NICD is subjected to various posttranslational modifications including ubiquitination. Here, we surprisingly found that NUMB proteins stabilize the intracellular domain of NOTCH1 receptor (N1ICD) by regulating the ubiquitin–proteasome machinery, which is independent of NUMB’s role in modulating endocytosis. BAP1, a deubiquitinating enzyme (DUB), was further identified as a positive N1ICD regulator, and NUMB facilitates the association between N1ICD and BAP1 to stabilize N1ICD. Intriguingly, BAP1 stabilizes N1ICD independent of its DUB activity but relying on the BRCA1-inhibiting function. BAP1 strengthens Notch signaling and maintains stem-like properties of cortical neural progenitor cells. Thus, NUMB enhances Notch signaling by regulating the ubiquitinating activity of the BAP1–BRCA1 complex.

scholarly journals High levels of Notch signaling down-regulate Numb and Numblike

2006 ◽  
Vol 175 (4) ◽  
pp. 535-540 ◽  
Author(s):  
Gavin Chapman ◽  
Lining Liu ◽  
Cecilia Sahlgren ◽  
Camilla Dahlqvist ◽  
Urban Lendahl
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Cultured Cells ◽  
Tumor Development ◽  
Proteasome Inhibitor Mg132 ◽  
Cell Fate Decisions ◽  
Protein Levels ◽  
Notch Intracellular Domain ◽  
Low Levels ◽  
Mediated Reduction

Inhibition of Notch signaling by Numb is critical for many cell fate decisions. In this study, we demonstrate a more complex relationship between Notch and the two vertebrate Numb homologues Numb and Numblike. Although Numb and Numblike at low levels of Notch signaling negatively regulated Notch, high levels of Notch signaling conversely led to a reduction of Numb and Numblike protein levels in cultured cells and in the developing chick central nervous system. The Notch intracellular domain but not the canonical Notch downstream proteins Hes 1 and Hey 1 caused a reduction of Numb and Numblike. The Notch-mediated reduction of Numblike required the PEST domain in the Numblike protein and was blocked by the proteasome inhibitor MG132. Collectively, these observations reveal a reciprocal negative regulation between Notch and Numb/Numblike, which may be of relevance for stabilizing asymmetric cell fate switches and for tumor development.

scholarly journals Cleaved Delta like 1 intracellular domain regulates neural development via Notch signal dependent and independent pathways

Development ◽  
2021 ◽  
Author(s):  
Yusuke Okubo ◽  
Fumiaki Ohtake ◽  
Katsuhide Igarashi ◽  
Yukuto Yasuhiko ◽  
Yoko Hirabayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Notch Signaling ◽  
Neural Development ◽  
Inhibition Mechanism ◽  
Independent Manner ◽  
Intracellular Domain ◽  
Neural Crest Stem Cells ◽  
Notch Intracellular Domain

Notch-Delta signaling regulates many developmental processes, including tissue homeostasis, and maintenance of stem cells. Upon interaction of juxtaposed cells via Notch and Delta proteins, intracellular domains of both transmembrane proteins are cleaved and translocate to the nucleus. Notch intracellular domain activates target gene expression; however, the role of the Delta intracellular domain remains elusive. Here, we show the biological function of Delta like 1 intracellular domain (D1ICD) by modulating its production. We find the sustained production of D1ICD abrogates cell proliferation but enhances neurogenesis in the developing dorsal root ganglia (DRG), whereas inhibition of D1ICD production promotes cell proliferation and gliogenesis. D1ICD acts as an integral component of lateral inhibition mechanism by inhibiting Notch activity. In addition, D1ICD promotes neurogenesis through a Notch signaling independent manner. We show that D1ICD binds to Erk1/2 in neural crest stem cells, and inhibits the phosphorylation of Erk1/2. In summary, our results indicate that D1ICD regulates DRG development via modulating not only Notch signaling but also the MAP kinase pathway.

scholarly journals Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0234101
Author(s):  
Jacob J. Crow ◽  
Allan R. Albig
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Regulatory Mechanism ◽  
Family Members ◽  
Regulatory Function ◽  
Specific Nature ◽  
Intracellular Domain ◽  
Notch Intracellular Domain ◽  
Highly Sensitive ◽  
Cellular Identity

Notch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch’s ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that the Notch 2 and 3 NICDs also exhibit dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Furthermore, our data suggested that Notch4 may also exhibit dimerization-induced signaling, although the amino acids required for Notch4 NICD dimerization appear to be different than those required for Notch 1, 2, and 3 NICD dimerization. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members’ SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.

scholarly journals Notch Intracellular Domain Plasmid Delivery via Poly(Lactic-Co-Glycolic Acid) Nanoparticles to Upregulate Notch Pathway Molecules

2021 ◽  
Vol 8 ◽  
Author(s):  
Victoria L. Messerschmidt ◽  
Uday Chintapula ◽  
Aneetta E. Kuriakose ◽  
Samantha Laboy ◽  
Thuy Thi Dang Truong ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Glycolic Acid ◽  
Umbilical Vein ◽  
Notch Pathway ◽  
Cellular Functions ◽  
Intracellular Domain ◽  
Downstream Signaling ◽  
Notch Intracellular Domain ◽  
Downstream Effects ◽  
Umbilical Vein Endothelial Cells

Notch signaling is a highly conserved signaling system that is required for embryonic development and regeneration of organs. When the signal is lost, maldevelopment occurs and leads to a lethal state. Delivering exogenous genetic materials encoding Notch into cells can reestablish downstream signaling and rescue cellular functions. In this study, we utilized the negatively charged and FDA approved polymer poly(lactic-co-glycolic acid) to encapsulate Notch Intracellular Domain-containing plasmid in nanoparticles. We show that primary human umbilical vein endothelial cells (HUVECs) readily uptake the nanoparticles with and without specific antibody targets. We demonstrated that our nanoparticles are non-toxic, stable over time, and compatible with blood. We further demonstrated that HUVECs could be successfully transfected with these nanoparticles in static and dynamic environments. Lastly, we elucidated that these nanoparticles could upregulate the downstream genes of Notch signaling, indicating that the payload was viable and successfully altered the genetic downstream effects.

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