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• W4310776066 abstract "Mutations in NOTCH3 underlie cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited cerebral small vessel disease. Two cleavages of NOTCH3 protein, at Asp80 and Asp121, were previously described in CADASIL pathological samples. Using monoclonal antibodies developed against a NOTCH3 neoepitope, we identified a third cleavage at Asp964 between an Asp-Pro sequence. We characterized the structural requirements for proteolysis at Asp964 and the vascular distribution of the cleavage event. A proteome-wide analysis was performed to find proteins that interact with the cleavage product. Finally, we investigated the biochemical determinants of this third cleavage event. Cleavage at Asp964 was critically dependent on the proline adjacent to the aspartate residue. In addition, the cleavage product was highly enriched in CADASIL brain tissue and localized to the media of degenerating arteries, where it deposited with the two additional NOTCH3 cleavage products. Recombinant NOTCH3 terminating at Asp964 was used to probe protein microarrays. We identified multiple molecules that bound to the cleaved NOTCH3 more than to uncleaved protein, suggesting that cleavage may alter the local protein interactome within disease-affected blood vessels. The cleavage of purified NOTCH3 protein at Asp964 in vitro was activated by reducing agents and NOTCH3 protein; cleavage was inhibited by specific dicarboxylic acids, as seen with cleavage at Asp80 and Asp121. Overall, we propose homologous redox-driven Asp-Pro cleavages and alterations in protein interactions as potential mechanisms in inherited small vessel disease; similarities in protein cleavage characteristics may indicate common biochemical modulators of pathological NOTCH3 processing. Mutations in NOTCH3 underlie cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited cerebral small vessel disease. Two cleavages of NOTCH3 protein, at Asp80 and Asp121, were previously described in CADASIL pathological samples. Using monoclonal antibodies developed against a NOTCH3 neoepitope, we identified a third cleavage at Asp964 between an Asp-Pro sequence. We characterized the structural requirements for proteolysis at Asp964 and the vascular distribution of the cleavage event. A proteome-wide analysis was performed to find proteins that interact with the cleavage product. Finally, we investigated the biochemical determinants of this third cleavage event. Cleavage at Asp964 was critically dependent on the proline adjacent to the aspartate residue. In addition, the cleavage product was highly enriched in CADASIL brain tissue and localized to the media of degenerating arteries, where it deposited with the two additional NOTCH3 cleavage products. Recombinant NOTCH3 terminating at Asp964 was used to probe protein microarrays. We identified multiple molecules that bound to the cleaved NOTCH3 more than to uncleaved protein, suggesting that cleavage may alter the local protein interactome within disease-affected blood vessels. The cleavage of purified NOTCH3 protein at Asp964 in vitro was activated by reducing agents and NOTCH3 protein; cleavage was inhibited by specific dicarboxylic acids, as seen with cleavage at Asp80 and Asp121. Overall, we propose homologous redox-driven Asp-Pro cleavages and alterations in protein interactions as potential mechanisms in inherited small vessel disease; similarities in protein cleavage characteristics may indicate common biochemical modulators of pathological NOTCH3 processing. NOTCH3 mutations result in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (1Joutel A. Corpechot C. Ducros A. Vahedi K. Chabriat H. Mouton P. et al.Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia.Nature. 1996; 383: 707-710Crossref PubMed Scopus (1725) Google Scholar), the most common inherited cerebral small vessel disease (2Chabriat H. Joutel A. Dichgans M. Tournier-Lasserve E. Bousser M.G. Cadasil.Lancet Neurol. 2009; 8: 643-653Abstract Full Text Full Text PDF PubMed Scopus (801) Google Scholar, 3Wang M.M. Cadasil.Handb Clin. Neurol. 2018; 148: 733-743Crossref PubMed Scopus (37) Google Scholar). The overwhelming majority of CADASIL mutations affect the number of cysteines in the NOTCH3 gene product (4Joutel A. Vahedi K. Corpechot C. Troesch A. Chabriat H. Vayssiere C. et al.Strong clustering and stereotyped nature of Notch3 mutations in CADASIL patients.Lancet. 1997; 350: 1511-1515Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 5Rutten J.W. Van Eijsden B.J. Duering M. Jouvent E. Opherk C. et al.The effect of NOTCH3 pathogenic variant position on CADASIL disease severity: NOTCH3 EGFr 1-6 pathogenic variant are associated with a more severe phenotype and lower survival compared with EGFr 7-34 pathogenic variant.Genet. Med. 2019; 21: 676-682Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), a characteristic that is capable of affecting one or more of the 102 disulfide bonds of the epidermal growth factor (EGF) domain array region of the extracellular domain of the protein. Pathological examination of CADASIL reveals marked accumulation of NOTCH3 protein (6Joutel A. Andreux F. Gaulis S. Domenga V. Cecillon M. Battail N. et al.The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients.J. Clin. Invest. 2000; 105: 597-605Crossref PubMed Scopus (474) Google Scholar) and other matrix proteins (7Monet-Lepretre M. Haddad I. Baron-Menguy C. Fouillot-Panchal M. Riani M. Domenga-Denier V. et al.Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.Brain. 2013; 136: 1830-1845Crossref PubMed Scopus (145) Google Scholar, 8Zhang X. Lee S.J. Young M.F. Wang M.M. The small leucine-rich proteoglycan BGN accumulates in CADASIL and binds to NOTCH3.Transl. Stroke Res. 2015; 6: 148-155Crossref PubMed Scopus (26) Google Scholar, 9Lee S.J. Zhang X. Wang M.M. Vascular accumulation of the small leucine-rich proteoglycan decorin in CADASIL.Neuroreport. 2014; 25: 1059-1063Crossref PubMed Scopus (11) Google Scholar, 10Dong H. Blaivas M. Wang M.M. Bidirectional encroachment of collagen into the tunica media in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.Brain Res. 2012; 1456: 64-71Crossref PubMed Scopus (27) Google Scholar, 11Young K.Z. Xu G. Keep S.G. Borjigin J. Wang M.M. Overlapping protein accumulation profiles of CADASIL and CAA: is there a common mechanism driving cerebral small-vessel disease?.Am. J. Pathol. 2021; 191: 1871-1887Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) in the smooth muscle layer of cerebral arteries. Protein accumulation is accompanied by severe vascular smooth muscle degeneration (12Ruchoux M.M. Maurage C.A. Cadasil: cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.J. Neuropathol. Exp. Neurol. 1997; 56: 947-964Crossref PubMed Scopus (246) Google Scholar). These genetic and pathological characteristics of CADASIL are consistent with a neomorphic mechanism of disease in which alterations of NOTCH3 tertiary structure trigger protein accumulation and vascular smooth muscle toxicity. The structural changes in NOTCH3 that occur in CADASIL have been revealed in studies that compare purified wildtype and mutant proteins (13Young K.Z. Rojas Ramírez C. Keep S.G. Gatti J.R. Lee S.J. Zhang X. et al.Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia.Commun. Biol. 2022; 5: 331Crossref PubMed Scopus (3) Google Scholar, 14Duering M. Karpinska A. Rosner S. Hopfner F. Zechmeister M. Peters N. et al.Co-aggregate formation of CADASIL-mutant NOTCH3: a single-particle analysis.Hum. Mol. Genet. 2011; 20: 3256-3265Crossref PubMed Scopus (77) Google Scholar). In contrast to normal NOTCH3, CADASIL mutants oligomerize, harbor free thiol groups, fragment readily, and are vulnerable to transreduction via cysteine containing NOTCH3 fragments (13Young K.Z. Rojas Ramírez C. Keep S.G. Gatti J.R. Lee S.J. Zhang X. et al.Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia.Commun. Biol. 2022; 5: 331Crossref PubMed Scopus (3) Google Scholar, 14Duering M. Karpinska A. Rosner S. Hopfner F. Zechmeister M. Peters N. et al.Co-aggregate formation of CADASIL-mutant NOTCH3: a single-particle analysis.Hum. Mol. Genet. 2011; 20: 3256-3265Crossref PubMed Scopus (77) Google Scholar, 15Cartee N.M.P. Lee S.J. Young K.Z. Zhang X. Wang M.M. Trans-reduction of cerebral small vessel disease proteins by notch-derived EGF-like sequences.Int. J. Mol. Sci. 2022; 23: 3671Crossref PubMed Scopus (3) Google Scholar, 16Wollenweber F.A. Hanecker P. Bayer-Karpinska A. Malik R. Bazner H. Moreton F. et al.Cysteine-sparing CADASIL mutations in NOTCH3 show proaggregatory properties in vitro.Stroke. 2015; 46: 786-792Crossref PubMed Scopus (37) Google Scholar). Additional modifications of NOTCH3 also include a series of post-translational alterations of NOTCH3: (a) redox-dependent changes in structure (17Zhang X. Lee S.J. Young K.Z. Josephson D.A. Geschwind M.D. Wang M.M. Latent NOTCH3 epitopes unmasked in CADASIL and regulated by protein redox state.Brain Res. 2014; 1583: 230-236Crossref PubMed Scopus (15) Google Scholar) and (b) pathological cleavage of the protein (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.J. Biol. Chem. 2020; 295: 1960-1972Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar). Two cleavages of NOTCH3 have been identified so far: cutting C terminal to Asp80 (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.J. Biol. Chem. 2020; 295: 1960-1972Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) and Asp121 (19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar). The cleavage of NOTCH3 at these locations is predicted to release two N-terminal NOTCH3 fragments (NTF and NTF2), which can bind to intact NOTCH3 ectodomain and transreduce a number of vascular matrix proteins (13Young K.Z. Rojas Ramírez C. Keep S.G. Gatti J.R. Lee S.J. Zhang X. et al.Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia.Commun. Biol. 2022; 5: 331Crossref PubMed Scopus (3) Google Scholar, 15Cartee N.M.P. Lee S.J. Young K.Z. Zhang X. Wang M.M. Trans-reduction of cerebral small vessel disease proteins by notch-derived EGF-like sequences.Int. J. Mol. Sci. 2022; 23: 3671Crossref PubMed Scopus (3) Google Scholar, 19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar). Both these cleavage events occur at Asp-Pro bonds situated between adjacent EGF-like domains (between repeats 1 and 2 for Asp80 and between 2 and 3 for Asp121). Four additional Asp-Pro sequences are found in human NOTCH3 ectodomain and have not yet been characterized. In the current work, we examined a third Asp-Pro site in NOTCH3 as a potential target of post-translational proteolysis in CADASIL. Using monoclonal antibodies that bind to the neoepitope generated by cleavage at Asp964, we describe the structural determinants and histological localization of this cleavage event. To understand potential consequences of the cleavage, we performed proteome-wide analysis of molecules capable of interacting with the product of Asp964 proteolysis. Finally, in vitro studies identified inhibitors and activators of cleavage, which suggest a mechanism that parallels that of cleavage at Asp80 and Asp121. There are six Asp-Pro sequences in the EGF-like repeat domain of human NOTCH3. Two sequences at positions 80 to 81 and 121 to 122 have been described before as disease-related sites of proteolysis in CADASIL (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.J. Biol. Chem. 2020; 295: 1960-1972Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar). Additional sites at positions 552 to 553, 854 to 855, 964 to 965, and 1086 to 1087 are located within the EGF-like repeats (Fig. 1A). Sites at positions 80 to 81, 121 to 122, 964 to 965, and 1086 to 1087 are situated between EGF-like repeats. Although all these Asp-Pro sequences may be candidate cleavage sites, this study focuses on sequences 964 to 965, which we hypothesized could be cleaved based on similarity to 80 to 81 and 121 to 122 (Fig. 1B). In prior work, we described generation of antibodies that recognize a neoepitope specific for the cleaved protein and that requires liberation of the C-terminal aspartic acid residue at the site of proteolysis (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.J. Biol. Chem. 2020; 295: 1960-1972Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar). A similar approach was taken in this work in which we generated neoepitope antibodies to detect cleavage of NOTCH3 at Asp964. Rabbit monoclonal antibodies were generated against a peptide found in NOTCH3 ending at Asp964: SCLCRPGYTGAHCQHEAD. Several clones were obtained with high specificity for the peptide. Clone 69B was selected for further characterization and used for all experiments described later. When tested against peptides on dot blots, 69B demonstrated a strong preference for sequences that ended precisely with the immunogen (Fig. 2A). The antibody did not recognize peptides with deletions of the terminal aspartate residue or peptides that contained addition of non-native residues after Asp964. There was very weak binding to peptides, which contained amino acid extensions beyond the aspartate of the immunogen. To test the requirement for the C-terminal aspartic acid residue for antibody recognition on immunoblots, we generated recombinant constructs that appended the target sequence to the C terminus of GFP. Additional constructs featured deletions and extensions of amino acids to the sequence ending in Asp964 of NOTCH3. When proteins from cells transfected with these constructs were evaluated by immunoblotting using 69B, only the protein ending precisely with Asp964 was strongly recognized (Fig. 2B). Cells transfected with selected GFP constructs in Figure 2B were fixed and stained using 69B. Though all transfectants stained equally for GFP, only cells receiving constructs that ended precisely with terminal Asp964 showed significant staining with 69B; deletion of the terminal Asp964 residue and addition of Pro965 to the recombinant GFP eliminated cell staining (Fig. 2C). We concluded that overall, 69B appears to strongly recognize protein ending in Asp964 of NOTCH3. Transfected 293 cells were analyzed to assess if NOTCH3 is cleaved in cells. Vector-transfected, wildtype full-length human NOTCH3, and R90C mutant–transfected cell lysates were incubated with 5 mM Tris(2-carboxyethyl)phosphine (TCEP) and analyzed by immunoblotting (Fig. 3A). Only NOTCH3-transfected cells expressed full-length NOTCH3 (red dot); this band did not react with 69B. Furthermore, using 69B, a faster migrating band appeared that was unique to NOTCH3-transfected cells. This finding was consistent with cleavage at Asp964 of the full-length wildtype and mutant protein in cell lysates. To characterize cleavage at Asp964 (which separates EGF-like repeats 24 and 25) in a purified system, we cloned complementary DNA (cDNA) sequences of NOTCH3 corresponding to EGF-like repeats 23 to 26 as fusions to the C terminus of mouse Fc. These recombinant clones were used to make stable 293 cell lines that secreted Fc-NOTCH3(23–26) protein into the conditioned media, from which pure recombinant NOTCH3 fragments were then prepared by Protein A-agarose affinity purification. Since Asp964 is located in the middle of the NOTCH3 sequences of Fc-NOTCH3(23–26), cleavage of the protein at Asp964 is predicted to liberate an Fc-containing fragment that is 8 kDa smaller than the intact protein. In the event of cleavage, the fragmented protein, but not the parent protein, is expected to react with 69B on immunoblots (Fig. 3B). Purified protein Fc-NOTCH3(23–26) from cell lines was separated by SDS-PAGE and demonstrated a major band reactive with anti-Fc antibodies at the expected size for uncleaved protein (Fig 3C; Fc band in red). This band was not reactive with 69B. But using 69B, we readily detected a smaller band at the size expected after Asp964 cleavage, which increased with time of incubation at 37 °C (Fig. 3C). Cleavage of NOTCH3 at other Asp-Pro sequences (positions 80–81 and 121–122) were shown to be enhanced by acidic pH (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.J. Biol. Chem. 2020; 295: 1960-1972Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar). We incubated Fc-NOTCH3(23–26) at five different pH values and analyzed the protein by immunoblotting using Fc and 69B to detect cleavage at Asp964. As shown in Figure 3, D and A, truncated 69B-reactive band was observed at all pH values, but there was significantly less at pH 9 (when considering the ratio of 69B-reactive protein to Fc-reactive undigested protein). Moderately acidic conditions (that previously enhanced cleavage at Asp-Pro positions 80–81 and 121–122 (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.J. Biol. Chem. 2020; 295: 1960-1972Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar)) did not enhance cleavage at Asp964, compared with neutral pH (Fig. 3D). Similarly, cleavage of NOTCH3 at Asp-Pro positions 80 to 81 and 121 to 122 is enhanced by protein reduction (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.J. Biol. Chem. 2020; 295: 1960-1972Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar). To test whether the same holds true for cleavage at Asp964, we incubated Fc-NOTCH3(23–26) with increasing doses of TCEP, which resulted in increased cleavage product when assessed by immunoblotting with 69B (Fig. 3E; far right panel). The reduction-sensitive cutting process was not affected by pretreatment with chaotropic agent guanidinium or with heat treatment, which did not reduce the amount of cleavage facilitated by TCEP (Fig. 3F; first and second sets of proteins). To test if Pro965 of the Asp-Pro cleavage sequence is required for cleavage, we created a series of point mutants of Fc-NOTCH3(23–26) in which NOTCH3 residue 965 was mutated to all other amino acids (Fig. 3G). Mutant constructs were transiently transfected into 293 cells, and Fc proteins from conditioned media were concentrated using Protein A beads before analysis by immunoblotting. Comparable amounts of Fc protein were recovered from the wildtype construct and all point mutants. As shown in Figure 3, H–J, the amount of cleaved protein that was reactive to 69B was highest for the wildtype protein containing the wildtype proline residue at position 965. Only trace amounts of 69B-reactive protein were found in all the mutants. This indicates that the proline that follows the Asp964 cleavage site is critical for protein fragmentation. We used immunohistochemistry to analyze the location of Asp964 within arteries in CADASIL brain samples (Fig. 4). When 69B was used on genetically defined CADASIL frontal lobe sections, deposits were found in both leptomeningeal and penetrating vessels of the white matter, particularly in thickened arteries in which vessels showed vascular media degeneration and intimal hyperplasia (regions between arrows and arrowheads; best seen in leptomeningeal arteries). In comparison, non-CADASIL control brains did not show consistent staining with 69B in cerebral arteries. The reactivity pattern for Asp964 cutting was similar to what we have previously described using antibodies that detect Asp80 and Asp121 cleavage (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.J. Biol. Chem. 2020; 295: 1960-1972Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar). Results represent analysis of 26 genetically defined CADASIL brains and 15 control brains without clinically apparent neurological disease. To better define the location of Asp964 cleavage in CADASIL samples, we stained serial sections with 69B (for Asp964 cleavage) and with antibodies UMI-F and 145H (for proteolysis at Asp80 and Asp121; Fig. 5). This revealed a markedly similar pattern in leptomeningeal arteries, where the borders between vascular layers are readily defined. Staining with all the antibodies appeared most intensely in the vascular media, defined by regions outside the internal elastic lamina that is seen in Miller staining of adjacent brain sections (Fig. 5; blue stain). The Asp964 cleavage was not as extensive as collagen deposition, which was found in both media and intima (Fig. 5; B-CHP for all collagens and M3F7 for type IV collagen). The 69B immunoreactivity also colocalized well with 2079 (NOTCH3 conformational antibody; (17Zhang X. Lee S.J. Young K.Z. Josephson D.A. Geschwind M.D. Wang M.M. Latent NOTCH3 epitopes unmasked in CADASIL and regulated by protein redox state.Brain Res. 2014; 1583: 230-236Crossref PubMed Scopus (15) Google Scholar)) in serial sections of the same artery (Fig. 5). Serial sections were also stained with 69B and mature smooth muscle markers ACTA2 and CNN1 (Fig. 5). In CADASIL, 69B reactivity in leptomeningeal arteries did not completely coincide with either smooth muscle marker; both ACTA2 and CNN1 was expressed strongly in the intima and in remnant balloon cells of the media. This agreed with prior work in which we showed redistribution of smooth muscle markers to the intima, whereas NOTCH3 remained localized to the media without preference to viable cells (20Gatti J.R. Zhang X. Korcari E. Lee S.J. Greenstone N. Dean J.G. et al.Redistribution of mature smooth muscle markers in brain arteries in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.Transl. Stroke Res. 2018; https://doi.org/10.1007/s12975-018-0643-xCrossref PubMed Scopus (9) Google Scholar). In control arteries, 69B reactivity was not appreciated above background, whereas ACTA2 and CNN1 were expressed strongly in the media. Numerous proteins interact with NOTCH3 and have been considered potential modulators of CADASIL pathogenesis (7Monet-Lepretre M. Haddad I. Baron-Menguy C. Fouillot-Panchal M. Riani M. Domenga-Denier V. et al.Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.Brain. 2013; 136: 1830-1845Crossref PubMed Scopus (145) Google Scholar, 8Zhang X. Lee S.J. Young M.F. Wang M.M. The small leucine-rich proteoglycan BGN accumulates in CADASIL and binds to NOTCH3.Transl. Stroke Res. 2015; 6: 148-155Crossref PubMed Scopus (26) Google Scholar, 9Lee S.J. Zhang X. Wang M.M. Vascular accumulation of the small leucine-rich proteoglycan decorin in CADASIL.Neuroreport. 2014; 25: 1059-1063Crossref PubMed Scopus (11) Google Scholar, 10Dong H. Blaivas M. Wang M.M. Bidirectional encroachment of collagen into the tunica media in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.Brain Res. 2012; 1456: 64-71Crossref PubMed Scopus (27) Google Scholar, 11Young K.Z. Xu G. Keep S.G. Borjigin J. Wang M.M. Overlapping protein accumulation profiles of CADASIL and CAA: is there a common mechanism driving cerebral small-vessel disease?.Am. J. Pathol. 2021; 191: 1871-1887Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 21Meng H. Zhang X. Lee S.J. Wang M.M. Von Willebrand factor inhibits mature smooth muscle gene expression through impairment of Notch signaling.PLoS One. 2013; 8: e75808Crossref PubMed Scopus (15) Google Scholar, 22Meng H. Zhang X. Lee S.J. Strickland D.K. Lawrence D.A. Wang M.M. Low density lipoprotein receptor-related protein-1 (LRP1) regulates thrombospondin-2 (TSP2) enhancement of Notch3 signaling.J. Biol. Chem. 2010; 285: 23047-23055Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 23Meng H. Zhang X. Hankenson K.D. Wang M.M. Thrombospondin 2 potentiates notch3/jagged1 signaling.J. Biol. Chem. 2009; 284: 7866-7874Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Having identified Asp964 cleavage in the vascular media of CADASIL, the principal site of pathology, we next explored whether the proteolytic product of this cleavage could interact with other proteins; moreover, we sought to test on a proteome-wide basis if some proteins preferentially interact with the cleavage product over the intact NOTCH3 protein. We applied purified protein fragments of Fc-NOTCH3(23–24), which includes two NOTCH3 EGF-like modules that terminate with Asp964 to protein microarrays that contain over 15,000 purified human proteins. A comparison group was generated by probing parallel protein microarrays with Fc-NOTCH3(23–26), in which four EGF-like modules include the uncleaved Asp-Pro sequence (Fig. 6A). Binding signals for all proteins on these microarrays revealed satisfactory reproducibility of binding levels. The experiment revealed that a subset of proteins were preferentially bound by Fc-NOTCH3(23–24) compared with Fc-NOTCH3(23–26). Another subset of proteins preferred binding to Fc-NOTCH3(23–26). An overview of the aggregate data is shown in Figure 6B, which shows a conventional volcano plot of the results, and in Figure 6C, we show a dot plot of all proteins tested by rank order of signal intensity. To explore whether individual proteins that bind to Asp964 could be classified according to biological pathway, we performed several analyses using established algorithms; a total of 261 proteins exhibited 1.5-fold increased binding to Fc-NOTCH3(23–24) compared with Fc-NOTCH3(23–26) (see Supporting information). The STRING algorithm (https://string-db.org) for exploration of protein interactions (Fig. 6D) shows potential interactions between proteins that bind to cleavage products; notable nodes in these networks include MAPK1 and PRKACG. Class analysis using the PANTHER algorithm (www.pantherdb.org) noted that the main protein class was transcriptional activators and metabolic enzymes (Fig. 6E). Binding proteins and cellular process (Fig. 6, F and G) were the most common categorizations of NOTCH3 truncation product–binding proteins. Of note, 18 groups of related proteins were identified as having preference for binding to protein terminating in Asp964 (Fig. 6H), which suggests that this NOTCH3 fragment may target a homologous protein domain common to each member of the class. On the other hand, most of these classes do not have significant homology, indicating that the targets of the Asp964-terminated NOTCH3 fragment are heterogeneous. Prior work has established experimental factors that regulated NOTCH3 Asp-Pro cleavage (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.J. Biol. Chem. 2020; 295: 1960-1972Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 19Zhang X. Lee S.J. Wang M.M. Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.Sci. Rep. 2021; 11: 17246Crossref PubMed Scopus (5) Google Scholar). To assess the effects of these factors on cleavage at Asp964, we analyzed purified Fc-NOTCH3(23–26) under defined conditions in vitro, using 69B immunoblotting to detect and quantify the efficiency of Asp964 proteolysis. In accordance with prior studies (18Young K.Z. Lee S.J. Zhang X. Cartee N.M.P. Torres M. Keep S.G. et al.NOTCH3 is non-enzymatically fragmente" @default.
• W4310776066 created "2022-12-17" @default.
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• W4310776066 date "2023-01-01" @default.
• W4310776066 modified "2023-09-29" @default.
• W4310776066 title "A midposition NOTCH3 truncation in inherited cerebral small vessel disease may affect the protein interactome" @default.
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