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

Young, Kelly Z.; Cartee, Naw May Pearl; Ivanova, Magdalena I.; Wang, Michael M.

doi:10.1016/j.expneurol.2020.113261

Cited by 6 publications

(8 citation statements)

References 49 publications

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“…Samples were boiled in sample buffer with or without reducing agents, as indicated. Western blots [ 24 ] were performed using standard methods on nitrocellulose membranes using the monoclonal antibodies followed by fluorescent anti-mouse secondary antibodies. For carboxamidomethyl-cysteine (CAM) residue detection, rabbit monoclonal antibodies 4E7 and 52H11 (manuscript in review) were used with anti-rabbit secondaries.…”

Section: Methodsmentioning

confidence: 99%

Binding of omeprazole to protein targets identified by monoclonal antibodies

Cartee

Wang

2020

PLoS ONE

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Omeprazole is the most commonly used proton pump inhibitor (PPI), a class of medications whose therapeutic mechanism of action involves formation of a disulfide linkage to cysteine residues in the H+/K+ ATPase pump on gastric secretory cells. Covalent linkage between the sole sulfur group of omeprazole and selected cysteine residues of the pump protein results in inhibition of acid secretion in the stomach, an effect that ameliorates gastroesophageal reflux and peptic ulcer disease. PPIs, though useful for specific conditions when used transiently, are associated with diverse untoward effects when used long term. The mechanisms underlying these potential off-target effects remain unclear. PPIs may, in fact, interact with noncanonical target proteins (non-pump molecules) resulting in unexpected pathophysiological effects, but few studies describe off-target PPI binding. Here, we describe successful cloning of monoclonal antibodies against protein-bound omeprazole. We developed and used monoclonal antibodies to characterize the protein target range of omeprazole, stability of omeprazole-bound proteins, and the involvement of cysteines in binding of omeprazole to targets. We demonstrate that a wide range of diverse proteins are targeted by omeprazole. Protein complexes, detected by Western blotting, are resistant to heat, detergents, and reducing agents. Reaction of omeprazole occurs with cysteine-free proteins, is not fully inhibited by cysteine alkylation, occurs at neutral pH, and induces protein multimerization. At least two other clinically used PPIs, rabeprazole and tenatoprazole, are capable of binding to proteins in a similar fashion. We conclude that omeprazole binds to multiple proteins and is capable of forming highly stable complexes that are not dependent on disulfide linkages between the drug and protein targets. Further studies made possible by these antibodies may shed light on whether PPI-protein complexes underlie off-target untoward effects of chronic PPI use.

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Section: Methodsmentioning

confidence: 99%

Binding of omeprazole to protein targets identified by monoclonal antibodies

Cartee

Wang

2020

PLoS ONE

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“…31,33 In addition, NTF, which localizes to pathologically affected vessels in CADASIL, has also been shown to undergo spontaneous thiol-mediated oligomerization in vitro. 34 NTF can form covalent conjugates with vascular catecholamines that enhance multimerization of the NOTCH3 fragmentation product. 34 More recent studies have highlighted the presence of NTF multimers in CADASIL tissue, implicating a role for NTF multimers in disease (unpublished data).…”

Section: Notch3 Assembliesmentioning

confidence: 99%

“…34 NTF can form covalent conjugates with vascular catecholamines that enhance multimerization of the NOTCH3 fragmentation product. 34 More recent studies have highlighted the presence of NTF multimers in CADASIL tissue, implicating a role for NTF multimers in disease (unpublished data).…”

Section: Notch3 Assembliesmentioning

confidence: 99%

“…For example, CADASIL vessels feature accumulation of the NOTCH3 ectodomain without simultaneous accumulation of the intracellular domain, NOTCH3 aggregates, abnormal NOTCH3 conformations enriched in disease, and NOTCH3 fragments, including an NTF. 19,23,34 In addition, other proteins, such as proteoglycans, collagens (COLs), and other extracellular matrix (ECM) proteins, have been shown to accumulate within the vessels as well, suggesting formation of extracellular protein complexes in disease. 15,35e37 The extracellular matrix is a network of highly cross-linked proteins that plays important roles in cell structure and support.…”

Section: Protein Accumulation and Signaling Dysregulation In Cadasilmentioning

confidence: 99%

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Overlapping Protein Accumulation Profiles of CADASIL and CAA

Young

Keep

et al. 2021

The American Journal of Pathology

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“…In a recent study, we demonstrated that a peptide corresponding to the first EGF-like repeat of NOTCH3 is capable of reducing disulfide bonds of the NOTCH3 ectodomain (NTF; NOTCH3 N-terminal fragment [ 15 , 16 ] [Young KZ, accepted for publication]. The pathological relevance of this phenomenon is underscored by the finding that NOTCH3 mutations cause the most commonly inherited cerebral small vessel disease (SVD), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

Trans-Reduction of Cerebral Small Vessel Disease Proteins by Notch-Derived EGF-like Sequences

Cartee

Lee

Young

et al. 2022

IJMS

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Cysteine oxidation states of extracellular proteins participate in functional regulation and in disease pathophysiology. In the most common inherited dementia, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), mutations in NOTCH3 that alter extracellular cysteine number have implicated NOTCH3 cysteine states as potential triggers of cerebral vascular smooth muscle cytopathology. In this report, we describe a novel property of the second EGF-like domain of NOTCH3: its capacity to alter the cysteine redox state of the NOTCH3 ectodomain. Synthetic peptides corresponding to this sequence (NOTCH3 N-terminal fragment 2, NTF2) readily reduce NOTCH3 N-terminal ectodomain polypeptides in a dose- and time-dependent fashion. Furthermore, NTF2 preferentially reduces regional domains of NOTCH3 with the highest intensity against EGF-like domains 12–15. This process requires cysteine residues of NTF2 and is also capable of targeting selected extracellular proteins that include TSP2 and CTSH. CADASIL mutations in NOTCH3 increase susceptibility to NTF2-facilitated reduction and to trans-reduction by NOTCH3 produced in cells. Moreover, NTF2 forms complexes with the NOTCH3 ectodomain, and cleaved NOTCH3 co-localizes with the NOTCH3 ectodomain in cerebral arteries of CADASIL patients. The potential for NTF2 to reduce vascular proteins and the enhanced preference for it to trans-reduce mutant NOTCH3 implicate a role for protein trans-reduction in cerebrovascular pathological states such as CADASIL.

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

Cited by 6 publications

References 49 publications

Binding of omeprazole to protein targets identified by monoclonal antibodies

Binding of omeprazole to protein targets identified by monoclonal antibodies

Overlapping Protein Accumulation Profiles of CADASIL and CAA

Trans-Reduction of Cerebral Small Vessel Disease Proteins by Notch-Derived EGF-like Sequences

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