scholarly journals Notch–Jagged signaling complex defined by an interaction mosaic

2021 ◽  
Vol 118 (30) ◽  
pp. e2102502118
Author(s):  
Matthieu R. Zeronian ◽  
Oleg Klykov ◽  
Júlia Portell i de Montserrat ◽  
Maria J. Konijnenberg ◽  
Anamika Gaur ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Regulatory Region ◽  
Interaction Network ◽  
Activation Mechanism ◽  
Signaling Complex ◽  
Notch1 Signaling ◽  
Rich Domain ◽  
X Ray Scattering ◽  
Epidermal Growth ◽  
Notch1 Receptor

The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network. The Notch1 membrane–proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8–EGF13 and Jagged1 C2–EGF3 activation sites as well as to two additional Jagged1 regions, EGF8–EGF11 and cysteine-rich domain. XL-MS and quantitative interaction experiments show that the three Notch1-binding sites on Jagged1 also engage intramolecularly. These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility, determined by small-angle X-ray scattering, support the formation of nonlinear architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal but engage directly to control Notch1 signaling, thereby redefining the Notch1–Jagged1 activation mechanism and indicating routes for therapeutic applications.

scholarly journals Notch-Jagged signaling complex defined by an interaction mosaic

2021 ◽  
Author(s):  
Matthieu R. Zeronian ◽  
Oleg Klykov ◽  
Júlia Portell i de Montserrat ◽  
Maria J. Konijnenberg ◽  
Anamika Gaur ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Regulatory Region ◽  
Activation Mechanism ◽  
Signaling Complex ◽  
Notch1 Signaling ◽  
Rich Domain ◽  
X Ray Scattering ◽  
Epidermal Growth ◽  
Notch1 Receptor ◽  
Cysteine Rich Domain

AbstractThe Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand Jagged1 to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical and structural techniques on the full extracellular complex and targeted sites,we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network.The Notch1 membrane-proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8-13 and Jagged1 C2-EGF3 activation sites, as well as to two additional Jagged1 regions, EGF 8-11 and cysteine-rich domain (CRD). XL-MS and quantitative interaction experiments show that the three Notch1 binding sites on Jagged1 also engage intramolecularly.These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility determined by small-angle X-ray scattering (SAXS), support the formation of backfolded architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal, but engage directly to control Notch1 signaling, thereby redefining the Notch1-Jagged1 activation mechanism and indicating new routes for therapeutic applications.

scholarly journals A Review on Notch Signaling and Colorectal Cancer

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1549
Author(s):  
Ashish Tyagi ◽  
Arun K. Sharma ◽  
Chendil Damodaran
Keyword(s):  
Colorectal Cancer ◽  
Monoclonal Antibodies ◽  
Notch Signaling ◽  
Regulatory Region ◽  
Severe Toxicity ◽  
Oncogenic Signaling ◽  
Notch1 Signaling ◽  
Notch Receptors ◽  
Notch1 Receptor

Colorectal cancer (CRC) has one of the highest mortality rates despite the advancement of treatment options. Aggressive CRC remains difficult to treat owing to the activation of oncogenic signaling pathways such as the Notch signaling pathway. The role of Notch receptors varies according to the difference in their structures; in particular, aberrant activation of Notch1 has been attributed to the severity of CRC. Notch1 activation in CRC is inhibited by small molecule inhibitors that target γ-secretase, an enzyme responsible for the third and last cleavage step of Notch receptors. γ-Secretase also produces the intracellular domain that finally carries out cellular functions by activating downstream effectors. However, most inhibitors block γ-secretase non-selectively and cause severe toxicity. Plant-source-derived small molecules, monoclonal antibodies, biological molecules (such as SiRNAs), and compounds targeting the Notch1 receptor itself or the downstream molecules such as HES1 are some of the options that are in advanced stages of clinical trials. The Negative Regulatory Region (NRR), which plays a central role in the transduction of Notch1 signaling in the event of ligand-dependent and ligand-independent Notch1 processing is also being targeted specifically by monoclonal antibodies (mAbs) to prevent aberrant Notch1 activation. In this review, we discuss the role of Notch1 in CRC, particularly its metastatic phenotype, and how mutations in Notch1, specifically in its NRR region, contribute to the aberrant activation of Notch1 signaling, which, in turn, contributes to CRC pathogenesis. We also discuss prevailing and emerging therapies that target the Notch1 receptor and the NRR region, and we highlight the potential of these therapies in abrogating Notch signaling and, thus, CRC development and progression.

scholarly journals Structural insights into the activation mechanism of dynamin-like EHD ATPases

2017 ◽  
Vol 114 (22) ◽  
pp. 5629-5634 ◽  
Author(s):  
Arthur Alves Melo ◽  
Balachandra G. Hegde ◽  
Claudio Shah ◽  
Elin Larsson ◽  
J. Mario Isas ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Cellular Membrane ◽  
Activation Mechanism ◽  
Gtpase Domain ◽  
Membrane Recruitment ◽  
Spin Resonance ◽  
Epidermal Growth ◽  
Electron Paramagnetic

Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50° rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

scholarly journals A modifier in the 129S2/SvPasCrl genome is responsible for the viability of Notch1[12f/12f] mice

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Shweta Varshney ◽  
Hua-Xing Wei ◽  
Frank Batista ◽  
Mohd Nauman ◽  
Subha Sundaram ◽  
...  
Keyword(s):  
Target Genes ◽  
Notch Pathway ◽  
X Ray ◽  
X Ray Crystallography ◽  
Notch1 Signaling ◽  
Notch Receptors ◽  
F1 Progeny ◽  
Epidermal Growth ◽  
Notch Ligands

Abstract Background Mouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. O-Fucose at this site has been shown by X-ray crystallography to be recognized by both DLL4 and JAG1 Notch ligands. We previously showed that a Notch1 Thr466Ala mutant exhibits very little ligand-induced NOTCH1 signaling in a reporter assay, whereas a Thr466Ser mutation enables the transfer of O-fucose and reverts the NOTCH1 signaling defect. We subsequently generated a mutant mouse with the Thr466Ala mutation termed Notch1[12f](Notch1tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile. Results We now report that after backcrossing to C57BL/6 J mice for 11–15 generations, few homozygous Notch1[12f/12f] embryos were born. Timed mating showed that embryonic lethality occurred by embryonic day (E) ~E11.5, somewhat delayed compared to mice lacking Notch1 or Pofut1 (the O-fucosyltransferase that adds O-fucose to Notch receptors), which die at ~E9.5. The phenotype of C57BL/6 J Notch1[12f/12f] embryos was milder than mutants affected by loss of a canonical Notch pathway member, but disorganized vasculogenesis in the yolk sac, delayed somitogenesis and development were characteristic. In situ hybridization of Notch target genes Uncx4.1 and Dll3 or western blot analysis of NOTCH1 cleavage did not reveal significant differences at E9.5. However, qRT-PCR of head cDNA showed increased expression of Dll3, Uncx4.1 and Notch1 in E9.5 Notch1[12f/12f] embryos. Sequencing of cDNA from Notch1[12f/12f] embryo heads and Southern analysis showed that the Notch1[12f] locus was intact following backcrossing. We therefore looked for evidence of modifying gene(s) by crossing C57BL/6 J Notch1 [12f/+] mice to 129S2/SvPasCrl mice. Intercrosses of the F1 progeny gave viable F2 Notch1[12f/12f] mice. Conclusion We conclude that the 129S2/SvPasCrl genome contains a dominant modifying gene that rescues the functions of NOTCH1[12f] in signaling. Identification of the modifying gene has the potential to illuminate novel factor(s) that promote Notch signaling when an O-fucose glycan is absent from EGF12 of NOTCH1.

scholarly journals Dynamin Forms a Src Kinase–sensitive Complex with Cbl and Regulates Podosomes and Osteoclast Activity

2005 ◽  
Vol 16 (7) ◽  
pp. 3301-3313 ◽  
Author(s):  
Angela Bruzzaniti ◽  
Lynn Neff ◽  
Archana Sanjay ◽  
William C. Horne ◽  
Pietro De Camilli ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Adaptor Protein ◽  
Molecular Signaling ◽  
Src Tyrosine Kinase ◽  
Signaling Complex ◽  
Rous Sarcoma ◽  
Rich Domain ◽  
Cytoskeleton Remodeling ◽  
Sarcoma Virus ◽  
And Migration

Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption.

scholarly journals Vinexin Forms a Signaling Complex with Sos and Modulates Epidermal Growth Factor-induced c-Jun N-terminal Kinase/Stress-activated Protein Kinase Activities

1999 ◽  
Vol 274 (50) ◽  
pp. 35933-35937 ◽  
Author(s):  
Masahiko Akamatsu ◽  
Shin-ichi Aota ◽  
Akira Suwa ◽  
Kazumitsu Ueda ◽  
Teruo Amachi ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Signaling Complex ◽  
Epidermal Growth

scholarly journals 20 YEARS OF LEPTIN: Insights into signaling assemblies of the leptin receptor

2014 ◽  
Vol 223 (1) ◽  
pp. T9-T23 ◽  
Author(s):  
Frank Peelman ◽  
Lennart Zabeau ◽  
Kedar Moharana ◽  
Savvas N Savvides ◽  
Jan Tavernier
Keyword(s):  
Electron Microscopy ◽  
Energy Homeostasis ◽  
Leptin Receptor ◽  
Structural Information ◽  
Cell Types ◽  
Activation Mechanism ◽  
Peripheral Cell ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering

Leptin plays a central role in the control of body weight and energy homeostasis, but is a pleiotropic cytokine with activities on many peripheral cell types. In this review, we discuss the interaction of leptin with its receptor, and focus on the structural and mechanistic aspects of the extracellular aspects of leptin receptor (LR) activation. We provide an extensive overview of all structural information that has been obtained for leptin and its receptor via X-ray crystallography, electron microscopy, small-angle X-ray scattering, homology modeling, and mutagenesis studies. The available knowledge is integrated into putative models toward a recapitulation of the LR activation mechanism.

scholarly journals Leukemia-Associated Mutations within the NOTCH1 Heterodimerization Domain Fall into at Least Two Distinct Mechanistic Classes

2006 ◽  
Vol 26 (12) ◽  
pp. 4642-4651 ◽  
Author(s):  
Michael J. Malecki ◽  
Cheryll Sanchez-Irizarry ◽  
Jennifer L. Mitchell ◽  
Gavin Histen ◽  
Mina L. Xu ◽  
...  
Keyword(s):  
Lymphoblastic Leukemia ◽  
Proteolytic Activation ◽  
Activating Mutations ◽  
Notch1 Signaling ◽  
Human T Cell ◽  
Secretase Activity ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Domain Stability ◽  
Increased Susceptibility ◽  
Notch1 Receptor

ABSTRACT The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and γ-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require γ-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.

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