Electrophilic and Drug-Induced Stimulation of NOTCH3 N-terminal Fragment Oligomerization in Cerebrovascular Pathology

Young, Kelly Z.; Cartee, Naw May Pearl; Lee, S. J.; Keep, Simon G.; Ivanova, Magdalena I.; Wang, Michael M.

doi:10.1007/s12975-021-00908-2

Cited by 3 publications

(4 citation statements)

References 31 publications

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“…Delta/Notch signaling has been shown to be dependent on H+/K + -ATPase for activation during L/R axis patterning in vertebrates and sea urchins ( Raya et al, 2004 ; Bessodes et al, 2012 ). Omeprazole has additionally been shown to induce oligomerization of the Notch3 N-terminal fragment, resulting in destabilization of the protein ( Young et al, 2021 ; 2022 ). We did not observe significant changes in the miRNA levels in response to DAPT or Omeprazole ( Supplementary Figure S2 ).…”

Section: Resultsmentioning

confidence: 99%

Transcription of microRNAs is regulated by developmental signaling pathways and transcription factors

Arnott,

Sampilo,

Song

2024

Front. Cell Dev. Biol.

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In early embryonic development, the cross-regulation of transcription factors and signaling pathways are critical in mediating developmental and physiological processes. Additionally, many studies have shown the importance of post-transcriptional regulation of signaling and network components mediated by microRNAs (miRNAs); however, how miRNAs are transcriptionally regulated is poorly understood. miRNAs are critical fine-tuners of many biological processes and their dysregulation leads to a variety of diseases and developmental defects. Previously, we have shown that miRNAs are dynamically expressed throughout sea urchin development, suggesting that miRNAs are likely to be under transcriptional regulation. Here, we used pharmacological inhibitors, genetic constructs, and loss-of-function reagents to assess the impact of key signaling pathways (Wnt, Nodal, MAPK, Sonic Hedgehog, Delta/Notch, VEGF, and BMP) and transcription factors (Alx1, Ets1/2, and Tbr) on the transcript levels of the evolutionarily conserved miR-1, miR-31, miR-92 and miR-124; the invertebrate-specific miR-71; and the echinoderm-specific miR-2002, miR-2007, and miR-2012. We also used computational methods to identify potential transcription factor binding sites of these miRNAs. Lists of binding motifs for transcription factors (TFs) were acquired from the MEME-Suite Motif Database and used as inputs for the algorithm FIMO (Find Individual Motif Occurrences), which detects short nucleotide motifs within larger sequences. Based on experimental data on miRNA expression in conjunction with bioinformatic predictions, we propose that the transcription factors Tbr, Alx1, and Ets1 regulate SpmiR-1, SpmiR-31, and SpmiR-71, respectively. We additionally observed significant effects on miRNA levels as a result of perturbations to Wnt, Nodal, MAPK, and Sonic Hedgehog signaling pathways, while no significant change on miRNA levels were observed with perturbations to Delta/Notch, VEGF, or BMP signaling pathways. Overall, this study provides insights into the transcriptional regulation of miRNAs by signaling pathways and transcription factors and contribute to our overall understanding of the genetic regulation of developmental processes.

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Section: Resultsmentioning

confidence: 99%

Transcription of microRNAs is regulated by developmental signaling pathways and transcription factors

Arnott,

Sampilo,

Song

2024

Front. Cell Dev. Biol.

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“…The primary antibodies for CD63 (H5C6) and NOTCH3 fragmentation product (UMI-F) were detected using oligonucleotide-labeled anti-mouse and anti-rabbit secondary antibodies, respectively. PLA was performed according to manufacturer’s brightfield PLA instructions (Sigma-Aldrich), except for increasing the amplification time to 5 hours [ 15 ].…”

Section: Methodsmentioning

confidence: 99%

“…The location of mRNA corresponding to CD63 and CTSH in CADASIL frontal lobe was assessed using a system designed by Advanced Cell Diagnostics. The protocol, provided by the manufacturer, involved hybridization of custom designed nucleic acid probes that were detected using multiple non-enzymatic amplification steps followed by chromogenic detection using an alkaline phosphatase-conjugated terminal probe as previously described [ 15 , 18 ]. Signal was documented using brightfield microscopy for magenta puncta.…”

Section: Methodsmentioning

confidence: 99%

“…Truncation of the protein between the first and second EGF-like repeats occurs primarily in the media [ 10 ]. Further, oligomeric complexes of NOTCH3 fragments, visualized by proximity ligation assays, reside in media [ 15 ]. Recently, we identified a second cleavage event in NOTCH3, which also occurs predominantly in vascular media [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

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Concentration of non-myocyte proteins in arterial media of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

et al. 2023

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The most common inherited cause of vascular dementia and stroke, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is caused by mutations in NOTCH3. Post-translationally altered NOTCH3 accumulates in the vascular media of CADASIL arteries in areas of the vessels that exhibit profound cellular degeneration. The identification of molecules that concentrate in the same location as pathological NOTCH3 may shed light on processes that drive cytopathology in CADASIL. We performed a two phase immunohistochemical screen of markers identified in the Human Protein Atlas to identify new proteins that accumulate in the vascular media in a pattern similar to pathological NOTCH3. In phase one, none of 16 smooth muscle cell (SMC) localized antigens exhibited NOTCH3-like patterns of expression; however, several exhibited disease-dependent patterns of expression, with antibodies directed against FAM124A, GZMM, MTFR1, and ST6GAL demonstrating higher expression in controls than CADASIL. In contrast, in phase two of the study that included 56 non-SMC markers, two proteins, CD63 and CTSH, localized to the same regions as pathological NOTCH3, which was verified by VesSeg, a customized algorithm that assigns relative location of antigens within the layers of the vessel. Proximity ligation assays support complex formation between NOTCH3 fragments and CD63 in degenerating CADASIL media. Interestingly, in normal mouse brain, the two novel CADASIL markers, CD63 and CTSH, are expressed in non-SMC vascular cells. The identification of new proteins that concentrate in CADASIL vascular media demonstrates the utility of querying publicly available protein databases in specific neurological diseases and uncovers unexpected, non-SMC origins of pathological antigens in small vessel disease.

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Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia

et al. 2022

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Cerebral small vessel disease (SVD) is a prevalent disease of aging and a major contributor to stroke and dementia. The most commonly inherited SVD, CADASIL, is caused by dominantly acting cysteine-altering mutations in NOTCH3. These mutations change the number of cysteines from an even to an odd number, but the impact of these alterations on NOTCH3 protein structure remain unclear. Here, we prepared wildtype and four mutant recombinant NOTCH3 protein fragments to analyze the impact of CADASIL mutations on oligomerization, thiol status, and protein stability. Using gel electrophoresis, tandem MS/MS, and collision-induced unfolding, we find that NOTCH3 mutant proteins feature increased amounts of inappropriate disulfide bridges, reduced cysteines, and structural instability. Presence of a second protein factor, an N-terminal fragment of NOTCH3 (NTF), is capable of further altering disulfide statuses of both wildtype and mutant proteins, leading to increased numbers of reduced cysteines and further destabilization of NOTCH3 structure. In sum, these studies identify specific cysteine residues alterations and quaternary structure induced by CADASIL mutations in NOTCH3; further, we validate that reductive factors alter the structure and stability of this small vessel disease protein.

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Electrophilic and Drug-Induced Stimulation of NOTCH3 N-terminal Fragment Oligomerization in Cerebrovascular Pathology

Cited by 3 publications

References 31 publications

Transcription of microRNAs is regulated by developmental signaling pathways and transcription factors

Transcription of microRNAs is regulated by developmental signaling pathways and transcription factors

Concentration of non-myocyte proteins in arterial media of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia

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