Effects of Diabetes Insipidus Mutations on Neurophysin Folding and Function

Abstract

Mechanisms underlying the pathogenicity of diabetes insipidus mutations were probed by studying their effects on the properties of bovine oxytocin-related neurophysin. The mutations G17V, ⌬E47, G57S, G57R, and C67STOP were each shown to have structural consequences that would diminish the conformational stability and folding efficiency of the precursors in which they were incorporated, and factors contributing to the origins of these property changes were identified. Effects of the mutations on dimerization of the folded proteins were similarly analyzed. The projected relative impact of the above mutations on precursor folding properties qualitatively parallels the reported relative severity of their effects on the biological handling of the human vasopressin precursor, but quantitative differences between thermodynamic effects and biological impact are noted and explored. The sole mutation for which no clear thermodynamic basis was found for its pathogenicity was 87STOP, suggesting that the region of the precursor deleted by this mutation plays a role in targeting independent from effects on folding, or participates in stabilizing interactions unique to the human vasopressin precursor.The hormone vasopressin is synthesized as part of a larger precursor that contains the vasopressin sequence at its amino terminus, followed by that of the disulfide-rich protein neurophysin (NP) 1 and a carboxyl-terminal glycopeptide known as copeptin (Ref. 1 and reviewed in Ref. 2). Oxytocin biosynthesis is similar (3), with the exception that the precursor lacks the copeptin segment, the function of which is unclear (4). The NP components of the two precursors ( VP NP) and ( Oxy NP) are highly homologous; the two bovine neurophysins have almost identical properties in vitro, including similar affinities for each of the two hormones (e.g. Ref.2). Following processing, which cleaves the hormones from NP, the mature hormones remain noncovalently bound to NP by forces basically analogous to those pre-existing within the precursor, leading to analogous NP self-association properties (5). The structure of NP, the nature of the noncovalent interactions between hormones and NP, and the effects of these interactions on NP properties have been extensively investigated in solution (e.g. Ref.2) and by crystallographic analysis (6, 7). Moreover, the central role of NP in vasopressin elaboration has become evident by the demonstration that familial neurogenic diabetes insipidus (FNDI), an autosomal dominant disease characterized by vasopressin deficiency, appears most frequently to be due to mutations in NP (e.g. Refs. 8 and 9) accompanied by loss of the proper targeting of the hormone to regulated neurosecretory granules (e.g. Refs. 10 and 11); retention of the mutated precursor in the endoplasmic reticulum is generally considered the cause of the ultimate death of the affected neurons (10).Many of the mutations involved in diabetes insipidus can be predicted, on the basis of what is already known, to exert their effects by directly or indir...

Clinical and molecular analysis of three families with autosomal dominant neurohypophyseal diabetes insipidus associated with a novel and recurrent mutations in the vasopressin-neurophysin II gene

“…Interference with the binding of AVP to neurophysin also appears to be a relatively common sub-motif in the pathogenesis of adFNDI, because mutations that replace this glycine with arginine or valine have been identified in two other adFNDI families (7,9). Other mutations identified in adFNDI also appear to impair binding by altering other residues in the binding pocket or the N-terminal part of the AVP moiety that normally fits in the pocket (1,20).…”

“…The mutations identified in adFNDI are varied in type and affect all moieties of the precursor except copeptin and the peptide links between them. However, all of them share the unique property of altering one or more amino acid residues known or reasonably presumed to be critical for proper folding and dimerization of the precursor (1,20). This distinct pattern has led to the hypothesis that adFNDI is caused by production of a mutant precursor that is retained in the endoplasmic reticulum (ER) of the neuron because it cannot assume the threedimensional conformation necessary to pass the ER quality-control mechanisms and transit through the Golgi to the neurosecretory granules where final processing, packaging and storage of the various components normally occur (1).…”

Familial neurohypophyseal diabetes insipidus due to a novel mutation in the arginine vasopressin–neurophysin II gene

“…Only a few mutations have been localized to the signal peptide or the AVP coding sequence, and none in the glycoprotein moiety. It is assumed that all known autosomal dominant mutations cause defective folding or dimerization of the precursor (13).…”

Autosomal dominant familial neurohypophyseal diabetes insipidus caused by a novel mutation in arginine-vasopressin gene in a Brazilian family

“…These fi ndings were reinforced by analyzes in vitro performed by Eubansks and cols. (20) demonstrating that the mutation G88S has weaker free energy of internal bonding between hormone and NPII than in the WT precursor.…”