S1 Cleavage Research Articles

Structures and deformation phases affecting the Diagorou-Darbani greenstone belt south of Téra (Liptako, West Niger, and West African Craton)

The present study focuses on the geological formations of the Diagorou-Darbani greenstone belt of southern Téra in the Niger Liptako (NE portion of the Man Shield of the West African Craton). The main objective of this study is to characterize the deformation phases that affected the Diagorou-Darbani greenstone belt to the south of Téra. The methodology implemented is based on a structural analysis using field data. These formations are affected by two major deformation phases, D1 and D2. Phase D1 comprises three episodes: D1a, D1b and D1c. The first episode (D1a) developed the S1 cleavage (NE-SW). This episode is linked to a NW-SE shortening (N130°). The second episode (D1b) is marked by S2 crenulation cleavage, micro-folding and the dextral reactivation of N45° shear zones. This episode is linked to an E-W shortening. The third episode (D1c) is highlighted by the NW-SE S3 fracture cleavage and the sinistral reactivation of the N45° shear zones. A N-S to NNE-SSW shortening was obtained for episode D1c. Phase D2 is essentially brittle. It favoured the development of submeridian fractures and conjugate WNW-ESE (sinistral) and ENE-WSW (dextral) shear fractures. This deformation is compatible with a generally E-W direction of shortening.

Pacific-Gondwana Permo-Triassic Orogenic Belt with Lesser Overprinted Cenozoic Deformation, Eastern Bird Head Peninsula, West Papua, Indonesia

The Paleozoic to Early Mesozoic Pacific-facing active margin of East Gondwana has been shown to extend into the Kemum Block of northern Bird Head Peninsula (western New Guinea), and was associated with Mid Paleozoic orogenic deformation and Devonian to Triassic silicic magmatism and foreland basin deposition. In the Mawi Bay area of the eastern Bird Head Peninsula, the (?) Permian Mawi Complex is a multiply-deformed unit with a pre-Late Triassic D1 deformation formed in an Andean back-arc setting associated with active margin tectonism. The D1 deformation is characterized by recumbent, isoclinal to tight, north-northeast facing folds with an axial planar S1 cleavage formed at a low metamorphic grade, and predated the unconformably overlying Upper Triassic - Middle Jurassic Tipuma Formation. South of the Mawi Complex, the Mesozoic-Paleogene succession of the northern Lengguru Fold Belt is affected by north-northwest trending folds, cleavage, and most of the succession dips moderately to gently to the west-southwest. This deformation is reflected in the underlying Mawi Complex by northwest-trending D2 structures that have folded the D1 folds, and have steeply inclined open to gentle folds, some polyclinal folds, and fault-related folds. The structures in the northern Lengguru Fold Belt may have influenced The Pliocene to Quaternary Central Bird Head Monocline associated with the Kemum Block uplifting in the northern Bird Head Peninsula

Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling.

Individuals with spinal cord injury (SCI) suffer from permanent disabilities such as severe motor, sensory and autonomic dysfunction. Neural stem cell transplantation has proven to be a potential strategy to promote regeneration of the spinal cord, since NSCs can produce neurotrophic growth factors and differentiate into mature neurons to reconstruct the injured site. However, it is necessary to optimize the differentiation of NSCs before transplantation to achieve a better regenerative outcome. Inhibition of Notch signaling leads to a transition from NSCs to neurons, while the underlying mechanism remains inadequately understood. Our results demonstrate that overexpression of fucosyltransferase 9 (Fut9), which is upregulated by Wnt4, promotes neuronal differentiation by suppressing the activation of Notch signaling through disruption of furin-like enzyme activity during S1 cleavage. In an in vivo study, Fut9-modified NSCs efficiently differentiates into neurons to promote functional and histological recovery after SCI. Our research provides insight into the mechanisms of Notch signaling and a potential treatment strategy for SCI.

Emplacement of a lamprophyric sheet swarm under ductile transpression in continental crust. Insights from a Variscan magmatic complex in the West Armorican belt, Western France

We present structural data from a Variscan magmatic complex of dominantly lamprophyric (kersantite) and associated felsic (microgranodiorite) sheets - the West Armorican swarm (WAS) - injected into Paleozoic metasedimentary series at the western extremity of a transpressional system in the Armorican belt, Western France. The comparative structural analysis of nearly 100 individual intrusive sheets and their host-rocks demonstrates that the WAS complex is a syntectonic magmatic structure which intruded during the onset of regional shortening via successive pulses alternating with three strain episodes (D1-D2-D3). The moderate/high dipping attitude of a great majority of intrusions, along with their prominent orientation parallel to the regional cleavage (S1), i.e. orthogonal to the direction of shortening, challenge theoretical predictions about magma-filled fractures in contractional settings. These apparently atypical features are reconciled in a kinematic model which emphasizes the influence of both host-rock planar anisotropies (S1 cleavage and D3 shear patterns) and structural inheritance on magma pathways, and instead hypothesizes the negligible role of the ambient stress field. Comparisons are attempted with syncollisional lamprophyre systems present in the European Variscides, and more especially in the SE Bohemian massif.

Multiscale profiling of protease activity in cancer

Diverse processes in cancer are mediated by enzymes, which most proximally exert their function through their activity. High-fidelity methods to profile enzyme activity are therefore critical to understanding and targeting the pathological roles of enzymes in cancer. Here, we present an integrated set of methods for measuring specific protease activities across scales, and deploy these methods to study treatment response in an autochthonous model of Alk-mutant lung cancer. We leverage multiplexed nanosensors and machine learning to analyze in vivo protease activity dynamics in lung cancer, identifying significant dysregulation that includes enhanced cleavage of a peptide, S1, which rapidly returns to healthy levels with targeted therapy. Through direct on-tissue localization of protease activity, we pinpoint S1 cleavage to the tumor vasculature. To link protease activity to cellular function, we design a high-throughput method to isolate and characterize proteolytically active cells, uncovering a pro-angiogenic phenotype in S1-cleaving cells. These methods provide a framework for functional, multiscale characterization of protease dysregulation in cancer.

Constraints on the nature of metamorphism in the Kalgoorlie gold camp (Yilgarn Craton, Western Australia) and implications for genetic models of gold mineralisation

The timing and nature of metamorphism in the Kalgoorlie gold camp (Yilgarn Craton, Western Australia) are key aspects to genetic models for gold mineralisation. Despite this, existing constraints on metamorphism are equivocal as to whether the Fimiston and Oroya Au–Te lodes formed pre- or post-peak regional metamorphism. Evaluation of an actinolite (S1)–chlorite (S2) crenulation cleavage in the Hannan’s Lake Serpentinite on the western margin of the Golden Mile Super Pit supports a syn-D1 timing for peak metamorphism and a post-peak metamorphic timing for the Fimiston and Oroya Au–Te lodes. These results support a key aspect of metamorphic models for gold mineralisation. If intrusion-related models are correct, they should not be premised on the argument that the Fimiston and Oroya Au–Te lodes are equivalent to other examples of metamorphosed, epithermal-type mineralisation in orogenic belts. KEY POINTS The definition of an S1 cleavage in the Hannan’s Lake Serpentinite by actinolite supports a syn-D1 timing (ca 2685–2680 Ma) for peak regional metamorphism (sub- to lower-greenschist facies) in the Kalgoorlie gold camp. A syn-D1 timing for peak regional metamorphism suggests that the D2 (ca 2675–2665 Ma) Fimiston and Oroya Au–Te lodes and D3 (ca 2650–2640 Ma) Mt Charlotte stockwork Au orebodies developed in post-peak metamorphic settings. A post-peak metamorphic timing for the Fimiston and Oroya Au–Te lodes suggests that these mineralisation types do not represent metamorphosed equivalents of epithermal Au–Te mineralisation.

Fluorescence-Based Assay For Measuring OMA1 Activity.

Mitochondrial fusion depends on proteolytic processing of the dynamin-related GTPase protein, OPA1, which is regulated by the mitochondrial zinc metalloproteinase, OMA1. Last year we published a report describing a novel approach to directly measure the enzymatic activity of OMA1 in whole cell lysates. This fluorescence-based reporter assay utilizes an eight amino acid peptide sequence referred to as the S1 cleavage site where OMA1 cleaves within OPA1 and is flanked by a fluorophore and quencher. In this chapter, we provide additional insight into the OMA1 activity assay.

The Late Neoproterozoic-Early Paleozoic basin of the western Argentine Precordillera: Insights from zircon U-Pb geochronology

In central-western Argentina, a belt including marine metasedimentary rocks and mafic-ultramafic bodies occurs throughout the western margin of the Precordillera. The belt is considered as the suture zone between the poorly known Chilenia terrane and the Cuyania terrane, part of the composite West Gondwana margin. It is assigned to the Late Neoproterozoic-Early Devonian based on fossil fauna and radiometric ages. In the southern sector of this belt, in the Peñasco area, two units crop out. The Peñasco Formation comprises metasandstone and metapelite spatially associated with mafic metavolcanic and metahyaloclastic rocks. Metagabbro bodies intrude the succession. The Garganta del León Formation consists of metasandstone and scarce metapelite where tractive and deformational sedimentary structures are preserved. Both units are affected by low-grade metamorphism, but the main foliation S1 and crenulation cleavage S2 are better developed in the Peñasco Formation rocks. U-Pb data on detrital zircon of two metasandstone samples from these units show a dominant detrital input from sources with 1.0–1.3 and 0.65–0.53 Ga ages. Detritus may come from reworked sedimentary units or from igneous/metamorphic complexes from the Cuyania terrane basement that was possibly exhumed in the Ocloyic orogen. A Gondwanan provenance for the Late Neoproterozoic-Cambrian population would also be plausible. A ca. 460 Ma zircon population in the Garganta del León Formation is interpreted to be derived from the Famatinian Arc. This would imply that the deposition of the sediment occurred after the collision of the Cuyania terrane against West Gondwana, and that the Ocloyic orogen acted as a barrier for detritus from the Famatinian Arc and other rocks further east.

Sidi M'Barek: a representative example of the Moroccan massive sulfide deposits

Abstract In addition to a Zn-rich lens representing the northern prolongation of the Draa Sfar volcanogenic massive sulfide deposit, the Sidi M'Barek deposit includes Cu-rich lenses in which gold is being recovered profitably as a by-product. The Cu-rich mineralization is composed of numerous structurally controlled veins whose morphology suits that of a lode rather than that of a stratiform orebody. Although the Cu-rich and Zn-rich orebodies are dominated by pyrrhotite, pyrrhotite is coarser grained in the Cu-rich lenses and mineralization is polyphase. Pyrrhotite is deformed and recrystallized; it is cross-cut and replaced by pyrite infilling veins. Pyrite also crystallizes along the S 1 cleavage and in pressure shadows around bioclasts in the disseminated ore. Chalcopyrite veins postdate both the pyrrhotite and pyrite phases. The concentration of Co and Au is higher in the Cu-rich lenses relative to the Zn-rich lenses, which have higher concentrations of Pb, Ag and As. Pyrite and pyrrhotite have distinct sulfur and lead isotopic signatures. We propose a polygenetic model in which a pre-existing syngenetic pyrrhotite-rich mineralization is remobilized in shear zones during a major ductile deformation event allowing deposition of pyrite. The shear zones were reopened during a late ductile–brittle deformation event allowing deposition of chalcopyrite.

The age of salinic deformation constrained by 40 Ar/ 39 Ar dating of multiple cleavage domains: Bathurst Supergroup, New Brunswick Appalachians

In the New Brunswick Appalachians, polydeformed felsic volcanic rocks of the Bathurst Supergroup record four cleavage-forming tectonic events, of which D1 and D2 record subduction-related underplating of buoyant elements of the Tetagouche backarc basin and subsequent collision between composite Laurentia and the Gander margin, respectively, during the Salinic orogenic cycle. Here we present an integrated approach to dating multiple cleavage domains in which we performed step heat 40Ar/39Ar analyses, microstructural observations and mineral-chemistry analysis on a suite of samples, as well as in situ40Ar/39Ar analysis on two samples from the suite with clear S1 and S2 cleavage relationships. We use this dataset to characterize the complex relationships between S1 and S2 white mica between samples at different structural settings across the supergroup. We refine the timing of S1 white mica growth, and hence M1-D1 of the Salinic cycle in the Bathurst Supergroup to ca. 452 to 437 Ma, and S2 white mica growth (also D2) to ca. 427 to 418 Ma. New and published data indicate local thermal resetting of white mica at ca. 411 Ma, which we interpret to indicate a buried intrusion, likely a component of the Central plutonic belt. Delineation of the two white mica age components, particularly within a single sample, was informed by both spatially-controlled analysis (in situ40Ar/39Ar), and the higher age precision of step heat 40Ar/39Ar analyses.

The thrust contact between the Canastra and Vazante groups in the Southern Brasília Belt: structural evolution, white mica crystallinity and implications for the Brasiliano orogeny

ABSTRACT: Two regional thrust-sheets of Neoproterozoic metasedimentary rocks occur in the Southern Brasília Belt, northwest Minas Gerais. The lower one comprises the Vazante Group, that is formed in the studied area, from base to top, by the Serra do Garrote (metapelites interlayered with carbonaceous phyllite), Serra do Poço Verde (beige to pink stromatolitic metadolomite with interlayered greenish slates), Morro do Calcário (gray stromatolitic metadolomite interlayered with gray slates) and Serra da Lapa (phyllite with dolarenitic lenses interlayered with slates) formations. The upper thrust sheet consists of the Canastra Group (Paracatu formation): laminated sericite phyllites and carbonaceous phyllites interlayered with quartzite. The Braziliano orogeny resulted in four phases of contractional deformation, associated with low-grade metamorphism. The first two (D1 and D2) are ductile, while the third and fourth ones (D3 and D4) are brittle-ductile. D1 developed a slaty S1 cleavage subparallel to the primary layering, with shallow to steep dips to NW. D2 developed a crenulation cleavage (S2) that dips moderately to NW and is associated with tight to isoclinal folds. D3 and D4 phases developed crenulations and open folds and kink bands. S3 dips steeply to NW, while S4 has moderate to steep dips to NE and SW. White mica crystallinity (Kübler index) measurements in metapelites indicate that both the Canastra and Vazante groups reached anchizone/epizone conditions, and metamorphic discontinuities along thrusts indicate that the peak of metamorphism is pre or syn-thrusting.

Restoration of Opa1-long isoform inhibits retinal injury-induced neurodegeneration.

Optic atrophy 1 (Opa1) is a critical factor that regulates fusion and other important functions of mitochondria. In mitochondrion, the N-terminal mitochondrial targeting sequence of Opa1 precursors is removed to generate Opa1 long isoforms (L-Opa1), which are further cleaved into short isoforms (S-Opa1). In the present study, we found that retinal ischemia-reperfusion (I/R) injury and intravitreal injection of carbonylcyanide m-chlorophenyl hydrazone (CCCP) both dramatically induced Opa1 cleavage and caused loss of L-Opa1. In cultured neuronal cells under hypoxia-reoxygenation (H/R) injury, similar changes for Opa1 were also observed. In contrast, restoration of L-Opa1 level by overexpression of S1 cleavage site deletion Opa1 splice 1 (Opa1-ΔS1) not only normalized the H/R-induced mitochondrial morphology changes, but also inhibited the H/R-induced apoptosis, necrosis, and the intracellular ATP loss. Furthermore, recovering L-Opa1 level in the I/R-injured retina by intravitreal injection of genipin or overexpression of Opa1-ΔS1 inhibited apoptosis, necrosis, cell loss in the ganglion cell layer and retinal thickness reduction. Together, our data demonstrated the loss of L-Opa1 is involved in the development of retinal I/R injury, indicating restoring L-Opa1 level may be considered as a therapeutic target for I/R injury-related diseases, at least for the retina. Key messages: Retinal ischemia-reperfusion (I/R) or hypoxia-reoxygenation (H/R) injury induces L-Opa1 loss. Opa1-ΔS1 overexpression inhibits H/R-induced L-Opa1 loss. Opa1-ΔS1 overexpression inhibits H/R-induced mitochondria morphology change. Opa1-ΔS1 and genipin inhibit retinal I/R injury-induced necroptosis. Opa1-ΔS1 and genipin inhibit retinal I/R injury-induced neurodegeneration.

Joint modeling of RNase footprint sequencing profiles for genome-wide inference of RNA structure.

Recent studies have revealed significant roles of RNA structure in almost every step of RNA processing, including transcription, splicing, transport and translation. RNase footprint sequencing (RNase-seq) has emerged to dissect RNA structures at the genome scale. However, it remains challenging to analyze RNase-seq data because of the issues of signal sparsity, variability and correlations among various RNases. We present a probabilistic framework, joint Poisson-gamma mixture (JPGM), for integrative modeling of multiple RNase-seq profiles. Combining JPGM with hidden Markov model allows genome-wide inference of RNA structures. We apply the joint modeling approach for inferring base pairing states on simulated data sets and RNase-seq profiles of the double-strand specific RNase V1 and single-strand specific RNase S1 in yeast. We demonstrate that joint analysis of V1 and S1 profiles outputs interpretable RNA structure states, while approaches that analyze each profile separately do not. The joint modeling approach predicts the structure states of all nucleotides in 3196 transcripts of yeast without compromising accuracy, while the simple thresholding approach misses 43% of the nucleotides. Furthermore, the posterior probabilities outputted by our model are able to resolve the structural ambiguity of ≈300 000 nucleotides with overlapping V1 and S1 cleavage sites. Our model also generates RNA accessibilities, which are associated with three-dimensional conformations.

Geochemical characterization of the Marinoan “Cap Carbonate” of the Niari-Nyanga Basin (Central Africa)

Despite recent works on the Schisto-calcaire Group of the West-Congolian Supergroup in Central Africa, Neoproterozoic Cap Carbonate have not been well documented in the Niari-Nyanga basin that extends from Gabon to Angola. The couplet “Cap Carbonate (SCIa) and Upper Diamictite”, that is a marker of the “Snowball Earth event” was studied in 5 sections from the Niari-Nyanga basin and in one section from the external zone of the Mayombe folded belt (MFB). Our paper presents a high-resolution data set based on results from petrography, geochemistry, mineralogy and stable isotope studies performed on Cap Carbonate samples. The estimation of the illite crystallinity indexes indicates different degrees of post-sedimentary transformations evolving from deep diagenesis in the syncline basin to an epimetamorphism in the external zone of the MFB. Thin-sections of rocks sampled in this latter section display metamorphic features materialized by recrystallization of minerals in a S1 cleavage plane. Despite such an observation, all our samples clearly show preservation of primary petrographic structures such as biolaminations, fenestrae and peloids. Geochemical data leads us to highlight that the Cap Carbonate is composed by a variety of carbonate types. These results support the fact that the Cap Carbonate was derived from a combination of biological and chemical processes: organomineralization and chemical precipitation from supersaturated solutions, with a negligible impact of post-depositional transformations. For all the studied sections, stable isotope profiles display a drop in δ13C values, from about −2.6‰ to −4‰, associated with δ18O values ranging from −6‰ to −10‰. Moreover, the similarity of the isotopic profiles of the studied Cap Carbonate (from the Niari-Nyanga basin and the MFB) and the close fit to the expected Neoproterozoic δ13C and δ18O seawater values constitute a strong indication of a weak impact of a diagenetic to epimetamorphic evolution on the carbon isotopic signature of the SCIa Cap Carbonate. In the light of this data set, our isotopic records must be considered as a reliable tracer of the Marinoan Glaciation within the West-Congolian Supergroup.

Structural Constraints and Numerical Simulation of Strain Localization in the Bendigo Goldfield, Victoria, Australia

Strain localization and fluid flow patterns adjacent to the massive quartz-hosted reefs of the Bendigo gold-field have been investigated using new structural observations, three-dimensional geometry of fold-fault relationships, and gold distribution. Quartz reef and vein formation in the Bendigo goldfield is associated with an initial E-W compression event that produced a penetrative upright cleavage and N-S–trending fold hinges. In the immediate hanging wall of quartz reefs with high gold grades, the S1 cleavage is locally deformed by an E-W–trending S2 cleavage that is attributed to a low-strain N-S–oriented compressive event. Further evidence of this low-strain event is provided by gently S plunging quartz-carbonate slickenline lineations on S1 cleavage surfaces and the reef margin surfaces and visible gold within the outer portions of the reef. The reefs are commonly situated on or immediately to the south of a left-flex (sinistral) change in strike of the axial surface of the folds, and the flexure occurs close to an anticlinal culmination defined by the double-plunging N-S–striking folds. The folds become deformed with meter to decimeter wavelengths, which generally affect the stiff units, in a disharmonic style. Accompanying this is a reactivation of reef margins by oblique slip and the formation of dilational sites and tensional vein arrays that provide the conduits for fluid flow and gold deposition. Numerical models that best simulate the observed distribution of strain, based upon the location of quartz reefs, involve NW-SE–oriented contraction with a significant sinistral strike-slip component. This contraction event has an overall N-S–sense orientation and is therefore consistent with deformation features in the immediate hanging wall of quartz reefs. The models also demonstrate a geometrical dependence on the localization of strain and, thus, the potential localization of fluid flow. Volume and shear strain increase with the flex angle, and variations in the magnitude of strain are also associated with variations in the strike angle of the flexure. This analysis highlights that the modification of preexisting fold structures and quartz veins by a low-strain event may enhance the localization of dilation sites within individual quartz reefs where there are subtle changes in the orientation of the reef margins. Such numerical modeling can also improve ranking of exploration targets based on fold geometry and provide a better understanding of fluid flow in hydrothermal mineral systems.

Ligand activation leads to regulated intramembrane proteolysis of fibroblast growth factor receptor 3

Fibroblast growth factor receptor 3 (FGFR3) is a major negative regulator of bone growth that inhibits the proliferation and differentiation of growth plate chondrocytes. Activating mutations of its c isoform cause dwarfism in humans; somatic mutations can drive oncogenic transformation in multiple myeloma and bladder cancer. How these distinct activities arise is not clear. FGFR3 was previously shown to undergo proteolytic cleavage in the bovine rib growth plate, but this was not explored further. Here, we show that FGF1 induces regulated intramembrane proteolysis (RIP) of FGFR3. The ectodomain is proteolytically cleaved (S1) in response to ligand-induced receptor activation, but unlike most RIP target proteins, it requires endocytosis and does not involve a metalloproteinase. S1 cleavage generates a C-terminal domain fragment that initially remains anchored in the membrane, is phosphorylated, and is spatially distinct from the intact receptor. Ectodomain cleavage is followed by intramembrane cleavage (S2) to generate a soluble intracellular domain that is released into the cytosol and can translocate to the nucleus. We identify the S1 cleavage site and show that γ-secretase mediates the S2 cleavage event. In this way we demonstrate a mechanism for the nuclear localization of FGFR3 in response to ligand activation, which may occur in both development and disease.

Evidence for Increased Exposure of the Notch1 Metalloprotease Cleavage Site upon Conversion to an Activated Conformation

Notch proteins are transmembrane receptors that normally adopt a resting state poised to undergo activating proteolysis upon ligand engagement. Receptor quiescence is maintained by three LIN12/Notch repeats (LNRs), which wrap around a heterodimerization domain (HD) divided by furin cleavage at site S1 during maturation. Ligand binding initiates signaling by inducing sensitivity of the HD to proteolysis at the regulated S2 cleavage site. Here, we used hydrogen exchange mass spectrometry to examine the solution dynamics of the Notch1 negative regulatory region in autoinhibited states before and after S1 cleavage, in a proteolytically sensitive "on" state, and in a complex with an inhibitory antibody. Conversion to the "on" state leads to accelerated deuteration in the S2 region and in nearby secondary structural elements within the HD. In contrast, complexation with the inhibitory antibody retards deuteration around the S2 site. Together, these studies reveal how S2 site exposure is promoted by receptor activation and suppressed by inhibitory antibodies.

NOTCH1 missense alleles associated with left ventricular outflow tract defects exhibit impaired receptor processing and defective EMT

Notch signaling is essential for proper cardiac development. We recently identified missense variants in the NOTCH1 receptor in patients with diverse left ventricular outflow tract (LVOT) malformations (NOTCH1 G661S and NOTCH1 A683T) that reduce ligand-induced Notch signaling. Here, we examine the molecular mechanisms that contribute to reduced signaling and perturbed development. We find that NOTCH1 A683T exhibits reduced S1 cleavage due to impaired trafficking through the endoplasmic reticulum (ER). This observation is consistent with improper localization of the variant receptor to the ER and decreased presentation at the cell surface. In contrast, the nearby mutation NOTCH1 G661S exhibits reduced cell-surface presentation in the absence of overt folding or trafficking defects. To examine the implications of these variants in disease pathogenesis, we investigated their effect on epithelial-to-mesenchymal transition (EMT), a critical process for development of the outflow tract. We find that these LVOT-associated NOTCH1 alleles can contribute to defective EMT in endothelial cell lines through impaired induction of Snail and Hes family members. These data represent the first description of a molecular mechanism underlying NOTCH1 mutations in individuals with LVOT malformations, and have important implications regarding the functional contribution of these alleles to a complex set of developmental defects.

Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor-like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum-Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors.

Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

BackgroundNotch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia.Principal FindingsThe X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity.Conclusions/SignificanceS1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or T-ALL-associated mutations lead to conformational changes of the NRR that permit metalloprotease cleavage.