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

Abstract

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.

A midposition NOTCH3 truncation in inherited cerebral small vessel disease may affect the protein interactome

“…The conditioned media of these clones (in OptiMEM; Invitrogen; catalog no. : 31985070) were collected, and multiple fractions were combined and purified by protein A agarose chromatography ( 15 ). Acidic elution of Fc protein was followed by immediate neutralization with neutral Tris; finally, protein was dialyzed with two exchanges of PBS.…”

“…For protein stimulation studies, Fc-NOTCH(23–26) was supplemented with Fc-NOTCH3(1–3) and mutants, which have been described before ( 13 , 15 ).…”

“…The structural changes in NOTCH3 that occur in CADASIL have been revealed in studies that compare purified wildtype and mutant proteins ( 13 , 14 ). In contrast to normal NOTCH3, CADASIL mutants oligomerize, harbor free thiol groups, fragment readily, and are vulnerable to transreduction via cysteine containing NOTCH3 fragments ( 13 , 14 , 15 , 16 ).…”

“…Two cleavages of NOTCH3 have been identified so far: cutting C terminal to Asp80 ( 18 ) and Asp121 ( 19 ). 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 ( 13 , 15 , 19 ). 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).…”

Molecular Mechanisms of Cerebrovascular Diseases

“…The genetic basis of the most prevalent inherited form of stroke, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), has also been identified: mutations in the protein NOTCH3. In this issue, Cartee et al [3] explored the effects of cleaved products of NOTCH3 on the redox state of several extracellular polypeptides. Their study supports a novel mechanism whereby disulfide bonds of disparate proteins may be ruptured by interactions with pathological NOTCH3.…”