Synthetic studies of the mutant proinsulin syndrome demonstrate correlation between folding efficiency and age of diabetes onset.

IF 2 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Balamurugan Dhayalan, Yen-Shan Chen, Chun-Lun Ni, Michael A Weiss
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引用次数: 0

Abstract

Purpose: Heterozygous mutations in the insulin gene can give rise to a monogenic diabetes syndrome due to toxic misfolding of the variant proinsulin in the endoplasmic reticulum (ER) of pancreatic β-cells. Clinical mutations are widely distributed in the sequence (86 amino acids). Misfolding induces chronic ER stress and interferes in trans with wildtype biosynthesis and secretion. In the present work we sought to study relative folding efficiencies of proinsulin variants in relation to age of disease onset.

Methods: To enable efficient preparation of non-foldable variants, we developed a four-segment native chemical-ligation scheme that exploits two native cysteines (CysB19 and CysA6; residues 19 and 71 in proinsulin) and an alanine in the connecting domain (AlaC20; residue 50). From N- to C terminus, the four segments have respective lengths 18, 31, 22 and 15 residues-convenient to "mix and match" native and variant synthetic segments as a platform technology.

Results: Folding of the reduced and unfolded polypeptides was investigated under three conditions: pH 10.6 (which promotes disulfide pairing as in the pharmaceutical manufacture of insulin) and pH 7.4 in the absence or presence of "foldase" protein disulfide isomerase. Whereas wild-type proinsulin efficiently folds to form a single dominant product (in accordance with classical studies), the clinical variants exhibited marked impairment, especially at neutral pH.

Conclusion: Among representative clinical variants, relative folding yields correlated with both degree of ER stress in cell culture and ages of clinical diabetes onset (neonatal, adolescence or adulthood). Implications for the native mechanism of nascent protein folding are discussed.

突变胰岛素原综合征的综合研究表明折叠效率与糖尿病发病年龄之间存在相关性。
目的:胰岛素基因的杂合突变可引起单基因糖尿病综合征,这是由于胰腺β细胞内质网(ER)中变异胰岛素原的毒性错误折叠。临床突变广泛分布于该序列(86个氨基酸)中。错误折叠诱导慢性内质网应激并干扰野生型生物合成和分泌。在目前的工作中,我们试图研究与疾病发病年龄相关的胰岛素原变异的相对折叠效率。方法:为了高效制备不可折叠变异,我们开发了一种四段天然化学连接方案,利用两种天然半胱氨酸(CysB19和CysA6;胰岛素原的19和71残基)和连接域的一个丙氨酸(AlaC20;残留50)。从N-端到C端,4个片段的长度分别为18、31、22和15个残基,方便作为平台技术对天然和变异合成片段进行“混搭”。结果:在三种条件下研究了还原和未折叠多肽的折叠:pH 10.6(如胰岛素制药中促进二硫配对)和pH 7.4在没有或存在“折叠酶”蛋白二硫异构酶的情况下。尽管野生型胰岛素原有效折叠形成单一优势产物(与经典研究一致),但临床变异表现出明显的损伤,特别是在中性ph下。结论:在具有代表性的临床变异中,相对折叠量与细胞培养中内质网应激程度和临床糖尿病发病年龄(新生儿、青春期或成年期)相关。讨论了新生蛋白折叠的内在机制。
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来源期刊
CiteScore
5.50
自引率
8.00%
发文量
131
审稿时长
>12 weeks
期刊介绍: The International Journal for Peptide Research & Therapeutics is an international, peer-reviewed journal focusing on issues, research, and integration of knowledge on the latest developments in peptide therapeutics. The Journal brings together in a single source the most exciting work in peptide research, including isolation, structural characterization, synthesis and biological activity of peptides, and thereby aids in the development of unifying concepts from diverse perspectives. The Journal invites substantial contributions in the following thematic areas: -New advances in peptide drug delivery systems. -Application of peptide therapeutics to specific diseases. -New advances in synthetic methods. -The development of new procedures for construction of peptide libraries and methodology for screening of such mixtures. -The use of peptides in the study of enzyme specificity and mechanism, receptor binding and antibody/antigen interactions -Applications of such techniques as chromatography, electrophoresis, NMR and X-ray crystallography, mass spectrometry.
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