Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

Sohma, Youhei; Hua, Qing; Liu, Ming; Phillips, Nelson B.; Whittaker, Jonathan; Whittaker, Linda; Ng, Aubree; Roberts, Charles T.; Arvan, Peter; Kent, Stephen B. H.; Weiss, Michael A.

doi:10.1074/jbc.m109.062992

Cited by 23 publications

(17 citation statements)

References 106 publications

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“…More work is still needed to understand how these point mutations affect proinsulin folding in the ER. However, it is worth noting that nearly all of these mutations are located at highly conserved residues in the preproinsulin molecule, some of which have already been shown to be important for local folding of the insulin moiety, which may help to align the B- and A-chains in order to facilitate native proinsulin disulfide pairing (Hua et al, 2006b; Liu et al, 2009, 2010c; Sohma et al, 2010; Weiss, 2009). Thus, even these mutations that do not directly alter proinsulin Cys residues are likely to perturb normal disulfide pairing of proinsulin.…”

Section: Lessons From Ins-gene Mutations: the Links Between Biologmentioning

confidence: 99%

INS-gene mutations: From genetics and beta cell biology to clinical disease

Liu

Sun

Cui

et al. 2015

Molecular Aspects of Medicine

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137

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A growing list of insulin gene mutations causing a new form of monogenic diabetes has drawn increasing attention over the past seven years. The mutations have been identified in the untranslated regions of the insulin gene as well as the coding sequence of preproinsulin including within the signal peptide, insulin B-chain, C-peptide, insulin A-chain, and the proteolytic cleavage sites both for signal peptidase and the prohormone convertases. These mutations affect a variety of different steps of insulin biosynthesis in pancreatic beta cells. Importantly, although many of these mutations cause proinsulin misfolding with early onset autosomal dominant diabetes, some of the mutant alleles appear to engage different cellular and molecular mechanisms that underlie beta cell failure and diabetes. In this article, we review the most recent advances in the field and discuss challenges as well as potential strategies to prevent/delay the development and progression of autosomal dominant diabetes caused by INS-gene mutations. It is worth noting that although diabetes caused by INS gene mutations is rare, increasing evidence suggests that defects in the pathway of insulin biosynthesis may also be involved in the progression of more common types of diabetes. Collectively, the (pre)proinsulin mutants provide insightful molecular models to better understand the pathogenesis of all forms of diabetes in which preproinsulin processing defects, proinsulin misfolding, and ER stress are involved.

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Section: Lessons From Ins-gene Mutations: the Links Between Biologmentioning

confidence: 99%

INS-gene mutations: From genetics and beta cell biology to clinical disease

Liu

Sun

Cui

et al. 2015

Molecular Aspects of Medicine

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111

137

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“…Indeed, previous studies have shown that mutations at either A7 or B19 cause improper folding of proinsulin in vivo (4). And a very recent study has shown that mutation of residue B5, which is in an evolutionarily conserved region in close proximity to the B7-A7 disulfide bridge, also impairs proinsulin folding (7). Taken together, the above studies illustrate how the genetics of PNDM has given insight into the biophysical mechanisms and evolutionary constraints of proinsulin folding.…”

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confidence: 86%

Unfolding the mechanisms of disease progression in permanent neonatal diabetes

Dhanvantari

2010

American Journal of Physiology-Endocrinology and Metabolism

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DIABETES MELLITUS can be described as a collection of metabolic diseases characterized by fasting hyperglycemia. Although the most prevalent forms of diabetes mellitus are the polygenic forms that result in ␤-cell destruction or dysfunction, there exist monogenic forms that are diagnosed within the first six months of life, termed neonatal diabetes mellitus. This is a rare condition, occurring in one out of every 400,000 to 500,000 births, and can be either transient or permanent. Through numerous genetic studies, the cause of permanent neonatal diabetes mellitus (PNDM) can be traced to mutations in genes encoding for a number of ␤-cell functions, including pancreatic and islet development, the insulin-secretory response to glucose, and insulin biosynthesis (1). Such studies have resulted in elegant examples of "bedside-to-bench-to-bedside" science, in which genetic mutations are first identified in human populations, examined for their molecular and cellular effects on ␤-cell function, and the results applied to developing personalized approaches to disease management. For example, mutations in the K ATP channel components Kir6.2 (KCNJ11) and SUR1 (ABCC8) have been identified as the major cause of PNDM (6). These mutations were found to result in a gain-offunction, which, when expressed in transgenic mice, recapitulated PNDM by impairing glucose-stimulated insulin secretion. As a result, patients harboring mutations in K ATP channels are treated with sulfonuylurea drugs instead of insulin. Additionally, as novel mutations in K ATP channels are identified, the molecular mechanism of the disease can be studied on a case-by-case basis both for new treatments and to reveal novel aspects of K ATP function (2).A similar story appears to be emerging with the discovery of mutations in the proinsulin gene (INS) in patients with PNDM. Ten heterozygous mutations in the human INS gene have been recently identified through linkage analysis of a group of patients in Chicago (8). Interestingly, the mutations occurred at biologically critical sites within the proinsulin molecule: the site of signal peptide cleavage, residues involved in disulfide bond formation, and at the C-peptide-A chain junction, a site for posttranslational processing. The sites of these mutations led to the hypothesis that the type of PNDM associated with mutations in the INS gene could result from the improper folding and/or processing of proinsulin.In this issue, Rajan et al. (5) have addressed this hypothesis by examining the subcellular processing, trafficking, and secretion of the mutant proinsulins found in the Chicago probands. However, instead of simply generating the mutations within human proinsulin and following trafficking patterns with traditional secretion assays and immunocytochemistry, they made use of an "all-in-one" construct with which they could follow subcellular trafficking in live cells, detect proinsulin processing, and directly measure glucose-stimulated insulin secretion. The construct design incorporated green fluorescent protei...

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“…The latter proteins lack a B1 residue and contain divergent side chains at positions B2-B5. In particular, IGF-specific residue Thr B5 is incompatible with efficient insulin chain combination (64) and is associated in vitro with formation of a competing 9 Although the present set of substitutions tested at B1 highlights the importance of its non-polar character, Arg B1 occurs as a rare variant in non-mammalian insulin sequences. An example is provided by the divergent insulin of the hagfish (103), a member of a primitive lineage of marine craniates that lack a vertebral column.…”

Section: B8mentioning

confidence: 98%

“…1-3) and reducing (lanes 4 -6) conditions. con (lanes 1 and 4) IGF-I disulfide isomer on redox-coupled refolding (64,98). Such divergent folding properties reflect co-evolution of specific IGF-binding proteins (64,88,89).…”

Section: B8mentioning

confidence: 99%

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Deciphering the Hidden Informational Content of Protein Sequences

Liu

Hua

Jia

et al. 2010

Journal of Biological Chemistry

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Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe B1 in placental mammals) is variably positioned within crystal structures and exhibits 1 H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1-B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with ␤-cell dysfunction and neonatal diabetes mellitus.The efficiency of protein folding poses a fundamental problem at the intersection of biophysics, cell biology, and medicine (1, 2). Because the existence of a unique and accessible ground state is unrepresentative of polypeptides as a class of heteropolymers, foldability is an evolved property of biological sequences (3). Current kinetic models envisage a funnel-shaped free-energy landscape, enabling multiple trajectories to the native state (4 -6). What distinguishes foldable from non-foldable sequences (7), and how are bottlenecks avoided (8 -10)? The salience of these questions has been reinforced by recognition of proteotoxicity as a general pathological mechanism underlying diverse diseases (11,12). Here, we describe a cryptic folding element in a protein that is dispensable once the native state has been reached.A model is provided by insulin, a globular protein central to the regulation of vertebrate metabolism (13). Its impaired biosynthesis causes ␤-cell dysfunction and permanent neonatalonset diabetes mellitus (DM) 4 (14 -17). The insulin gene encodes a single-chain precursor, preproinsulin (Fig. 1A, top) (18). A signal peptide (gray bar) is cleaved on translocation into the endoplasmic reticulum (ER) to yield proinsulin. The precursor contains successive sequence motifs, defining B, C, and A domains (blue, black, and red , respectively, in Fig. 1A) (19). Whereas the translocated polypeptide is reduced and unfolded, oxidative folding in the ER yields a well organized A-B (insulinlike) core and disordered C-domain (dashed black segment in Fig. 1B) (20 -26). Folding is ...

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Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

Cited by 23 publications

References 106 publications

INS-gene mutations: From genetics and beta cell biology to clinical disease

INS-gene mutations: From genetics and beta cell biology to clinical disease

Unfolding the mechanisms of disease progression in permanent neonatal diabetes

Deciphering the Hidden Informational Content of Protein Sequences

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