Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.

Cerebrovascular pathology at the biochemical level has been informed by the study of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a vascular disorder caused by NOTCH3 mutations. Previous work in CADASIL described N-terminal proteolysis of NOTCH3 generated by specific non-enzymatic cleavage of the first Asp-Pro sequence of the protein. Here, we investigated whether the second Asp-Pro peptide bond (residues 121-122) of NOTCH3 is cleaved in CADASIL. Monospecific antibodies were generated that recognize the neo-epitope predicted to be generated by cleavage after Asp121. These antibodies were used to localize cleavage events at Asp121 in post-mortem CADASIL and control brain tissue and to investigate factors that regulate cleavage at Asp121. We report that cleavage at Asp121 occurs at a high level in the arterial media of CADASIL cerebral arteries. Leptomeningeal arteries demonstrated substantially more cleavage product than penetrating arteries in the white matter, and control vessels harbored only a small amount of cleaved NOTCH3. Proteolysis at Asp121 occurred in purified preparations of NOTCH3 ectodomain, was increased by acidic pH and reductive conditions, and required native protein conformation for cleavage. Increasing the concentration of NOTCH3 EGF-like domain protein elevated the level of proteolysis. On the other hand, several polyanionic chemicals potently blocked cleavage at Asp121. These studies demonstrate that the NOTCH3 protein in CADASIL is cleaved in multiple locations at labile Asp-Pro peptide bonds. As such, chronic brain vascular disease, like other neurodegenerative conditions, features proteolysis of pathological proteins at multiple sites which may generate small pathological peptides.

Contribution of "Omic" Studies to the Understanding of Cadasil. A Systematic Review.

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the epidermal growth factor (EGF)-like repeat domain, causing protein misfolding and aggregation. The main symptoms are migraines, psychiatric disorders, recurrent strokes, and dementia. Omic technologies allow the massive study of different molecules for understanding diseases in a non-biased manner or even for discovering targets and their possible treatments. We analyzed the progress in understanding CADASIL that has been made possible by omics sciences. For this purpose, we included studies that focused on CADASIL and used omics techniques, searching bibliographic resources, such as PubMed. We excluded studies with other phenotypes, such as migraine or leukodystrophies. A total of 18 articles were reviewed. Due to the high prevalence of NOTCH3 mutations considered pathogenic to date in genomic repositories, one can ask whether all of them produce CADASIL, different degrees of the disease, or whether they are just a risk factor for small vessel disease. Besides, proteomics and transcriptomics studies found that the molecules that are significantly altered in CADASIL are mainly related to cell adhesion, the cytoskeleton or extracellular matrix components, misfolding control, autophagia, angiogenesis, or the transforming growth factor ß (TGFß) signaling pathway. The omics studies performed on CADASIL have been useful for understanding the biological mechanisms and could be key factors for finding potential drug targets.

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion.

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.

A Novel Mutation Outside of the EGFr Encoding Exons of NOTCH3 Gene in a Chinese with CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary cerebral small vascular disease caused by the mutations of the NOTCH3 gene. The NOTCH3 gene consists of 33 exons. The pathogenic mutations of the NOTCH3 gene in CADASIL are located in 2-24 exons coding for the 34 EGFr (epidermal growth factor-like repeat) domains. The classical clinical manifestations are recurrent TIA or ischaemic stroke, migraine, cognitive disorder and affective disorder. The deposition of granular osmiophilic material (GOM) in the vascular wall is considered as a hallmark of the disease. METHODS: Here, we report a rare pathogenic mutation on exon 29 of the NOTCH3 gene in a Chinese family. Clinical data for the proband and available relatives is collected. Mutation analysis of the NOTCH3 gene was performed by screening the entire 33 exons in this family and 200 normal controls. A complete imaging evaluation and skin biopsy were performed on the proband. RESULTS: We identified a novel R1761H (c.5282G>A) mutation. The same mutation was not founded in 200 normal controls. The proband had recurrent stroke, depression, cognitive decline and cerebral lobe hemorrhage. Cranial MRI showed white matter lesions and multiple infarction. Susceptibility weighted imaging revealed numerous microbleeds.Most importantly, the deposition of GOM was found in the proband. CONCLUSION: 33 exons of NOTCH3 gene should be performed for individuals with a convincing CADASIL phenotype and positive family history.

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

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.

Thiol-mediated and catecholamine-enhanced multimerization of a cerebrovascular disease enriched fragment of NOTCH3.

Cerebral small vessel disease is a common condition linked to dementia and stroke. As an age-dependent brain pathology, cerebral SVD may share molecular processes with core neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Many neurodegenerative diseases feature abnormal protein accumulation and aberrant protein folding, resulting in multimerization of specific proteins. We investigated if a small NOTCH3 N-terminal fragment (NTF) that co-registers with pathologically affected cells in the inherited SVD, CADASIL, is capable of multimerization. We also characterized endogenous small molecule vascular enhancers and inhibitors of multimerization. NTF multimerizes spontaneously and also forms conjugates with vascular catecholamines, including dopamine and norepinephrine, which avidly promote multimerization of the protein. Inhibition of catecholamine-dependent multimerization by vitamin C and reversal by reducing agents implicate an essential role of oxidation in NTF multimerization. Antibodies that react with degenerating arteries in CADASIL tissue preferentially bind to multimerized forms of NTF. These studies suggest that multimerization of proteins in the aging brain is not restricted to neuronal molecules and may participate in age-dependent vascular pathology.

A new NOTCH3 damaging variant in a thrombocytopenia family of Miao ethnic group.

BACKGROUND: Pediatric inherited thrombocytopenia, also known as a deficiency of platelets in children, is caused by genetic factors and it is hard to obtain an effective treatment. Thus, it is necessary to identify the possible genetic variants that are responsible for thrombocytopenia. METHODS: Whole exome sequencing was used to detect genetic variants in two members of a thrombocytopenia family of Miao ethnic group. Multiple in silico analyses were performed to evaluate the effects of the novel missense variants. RESULTS: Finally, a novel variant (chr19: g.15170364G>A) in the NOTCH3 gene was found, as confirmed with Sanger sequencing, which could result in a R1694Q substitution in the protein. This variant was consistently suggested to be damaging by sift (Sorting Tolerant From Intolerant; http://sift.jcvi.org), polyphen (Polymorphism Phenotyping, version 2.0; http://genetics.bwh.harvard.edu/pph2) and mutationtaster (http://www.mutationtaster.org) software. By building the 3D model of the key region of NOTCH3 protein and performing the structure simulation, we found that (i) this variant affected the 3D structure model with a root-mean-square deviation = 0.46 between wild-type and mutant type; (ii) this variant caused the protein to reduce the solvent accessible surface area by 421 Å2 ; and (iii) compared to the wild-type protein, the mutant protein had two less amino acids to maintain protein stability. CONCLUSIONS: A novel damaging variant in the NOTCH3 gene was identified in a thrombocytopenia family with respect to decreasing the stability of NOTCH3, which may help with the prognosis and therapy of inherited thrombocytopenia.

Genetic diagnosis of CADASIL in three Hong Kong Chinese patients: A novel mutation within the intracellular domain of NOTCH3.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset hereditary stroke syndrome characterized by recurrent stroke and progressive cognitive impairment caused by NOTCH3 mutations. We report here the clinical and molecular findings of three unrelated Hong Kong Chinese families with CADASIL syndrome. Sanger sequencing of genomic DNA revealed a novel heterozygous variant NM_000435.2(NOTCH3):c.[5903_5904insATAA];[5903_5904=] NP_000426.2:p.(Asp1969∗);(Asp1969=) and two previously reported heterozygous mutations NM_000435.2(NOTCH3):c.[328C>T];[328C=] NP_000426.2:p.[(Arg110Cys)];[(Arg110=)] and NM_000435.2(NOTCH3):c.[580T>A];[580T=] NP_000426.2:p.(Cys194Ser);(Cys194=) in the three families respectively. Molecular basis of CADASIL in these three patients were further established. Genetic analysis provides a reliable method for confirming the diagnosis of CADASIL and enables proper genetic counseling and cascade testing.

[CADASIL with cysteine-sparing NOTCH3 mutation manifesting as dissociated progression between cognitive impairment and brain image findings in 3 years: A case report].

A 55-year-old man with no history of stroke or migraine presented to the clinic with cognitive impairment and depression that had been experiencing for two years. Neurological examination showed bilateral pyramidal signs, and impairments in cognition and attention. Brain MRI revealed multiple lacunar lesions and microbleeds in the deep cerebral white matter, subcortical regions, and brainstem, as well as diffuse white matter hyperintensities without anterior temporal pole involvement. Cerebral single-photon emission computed tomography (SPECT) revealed bilateral hypoperfusion in the basal ganglia. Gene analysis revealed an arginine-to-proline missense mutation in the NOTCH3 gene at codon 75. The patient was administered lomerizine (10 mg/day), but the patient's cognitive impairment and cerebral atrophy continued to worsen. Follow-up testing with MRI three years after his initial diagnosis revealed similar lacunar infarctions, cerebral microbleeds, and diffuse white matter hyperintensities to those observed three years earlier. However, MRI scans revealed signs of increased cerebral blood flow. Together, these findings suggest that the patient's cognitive impairments may have been caused by pathogenesis in the cerebral cortex.

Identification of Novel Breast Cancer Risk Loci.

It has been estimated that >1,000 genetic loci have yet to be identified for breast cancer risk. Here we report the first study utilizing targeted next-generation sequencing to identify single-nucleotide polymorphisms (SNP) associated with breast cancer risk. Targeted sequencing of 283 genes was performed in 240 women with early-onset breast cancer (≤40 years) or a family history of breast and/or ovarian cancer. Common coding variants with minor allele frequencies (MAF) >1% that were identified were presumed initially to be SNPs, but further database inspections revealed variants had MAF of ≤1% in the general population. Through prioritization and stringent selection criteria, we selected 24 SNPs for further genotyping in 1,516 breast cancer cases and 1,189 noncancer controls. Overall, we identified the JAK2 SNP rs56118985 to be significantly associated with overall breast cancer risk. Subtype analysis performed for patient subgroups defined by ER, PR, and HER2 status suggested additional associations of the NOTCH3 SNP rs200504060 and the HIF1A SNP rs142179458 with breast cancer risk. In silico analysis indicated that coding amino acids encoded at these three SNP sites were conserved evolutionarily and associated with decreased protein stability, suggesting a likely impact on protein function. Our results offer proof of concept for identifying novel cancer risk loci from next-generation sequencing data, with iterative data analysis from targeted, whole-exome, or whole-genome sequencing a wellspring to identify new SNPs associated with cancer risk. Cancer Res; 77(19); 5428-37. ©2017 AACR.

A novel NOTCH3 mutation identified in patients with oral cancer by whole exome sequencing.

Oral cancer is a serious disease caused by environmental factors and/or susceptible genes. In the present study, in order to identify useful genetic biomarkers for cancer prediction and prevention, and for personalized treatment, we detected somatic mutations in 5 pairs of oral cancer tissues and blood samples using whole exome sequencing (WES). Finally, we confirmed a novel nonsense single-nucleotide polymorphism (SNP; chr19:15288426A>C) in the NOTCH3 gene with sanger sequencing, which resulted in a N1438T mutation in the protein sequence. Using multiple in silico analyses, this variant was found to mildly damaging effects on the NOTCH3 gene, which was supported by the results from analyses using PANTHER, SNAP and SNPs&GO. However, further analysis using Mutation Taster revealed that this SNP had a probability of 0.9997 to be 'disease causing'. In addition, we performed 3D structure simulation analysis and the results suggested that this variant had little effect on the solubility and hydrophobicity of the protein and thus on its function; however, it decreased the stability of the protein by increasing the total energy following minimization (-1,051.39 kcal/mol for the mutant and -1,229.84 kcal/mol for the native) and decreasing one stabilizing residue of the protein. Less stability of the N1438T mutant was also supported by analysis using I-Mutant with a DDG value of -1.67. Overall, the present study identified and confirmed a novel mutation in the NOTCH3 gene, which may decrease the stability of NOTCH3, and may thus prove to be helpful in cancer prognosis.

Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.