Correcting temperature-sensitive protein folding defects.

Brown, C. Randell; Hong-Brown, Ly Q.; Welch, William J.

doi:10.1172/jci119302

Cited by 173 publications

(114 citation statements)

References 34 publications

(30 reference statements)

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“…The addition of glycerol and incubation at a reduced temperature are conditions that promote, in cultured cells, the correct processing of membrane proteins trapped in the ER or Golgi. [54][55][56] In an initial experiment, the effects of glycerol alone, incubation at 28°C alone, or glycerol and 28°C in combination on wild-type BSEP, R948C (c.2842CϾT), and E297G (c. 890AϾG) were assessed. Fig.…”

Section: Resultsmentioning

confidence: 99%

Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing

et al. 2008

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The gene encoding the human bile salt export pump (BSEP), ABCB11, is mutated in several forms of intrahepatic cholestasis. Here we classified the majority (63) of known ABCB11 missense mutations and 21 single-nucleotide polymorphisms (SNPs) to determine whether they caused abnormal ABCB11 pre-messenger RNA splicing, abnormal processing of BSEP protein, or alterations in BSEP protein function. Using an in vitro minigene system to analyze splicing events, we found reduced wild-type splicing for 20 mutations/SNPs, with normal mRNA levels reduced to 5% or less in eight cases. The common ABCB11 missense mutation encoding D482G enhanced aberrant splicing, whereas the common SNP A1028A promoted exon skipping. Addition of exogenous splicing factors modulated several splicing defects. Of the mutants expressed in vitro in CHO-K1 cells, most appeared to be retained in the endoplasmic reticulum and degraded. A minority had BSEP levels similar to wild-type. The SNP variant A444 had reduced levels of protein compared with V444. Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Taurocholate transport by two assessed mutants, N490D and A570T, was reduced compared with wild-type. Conclusion: This work is a comprehensive analysis of 80% of ABCB11 missense mutations and singlenucleotide polymorphisms at pre-mRNA splicing and protein processing/functional levels. We show that aberrant pre-mRNA splicing occurs in a considerable number of cases, leading to reduced levels of normal mRNA. Thus, primary defects at either the protein or the mRNA level (or both) contribute significantly to BSEP deficiency. These results will help to develop mutation-specific therapies for children and adults suffering from intrahepatic cholestasis due to BSEP deficiency. (HEPATOLOGY 2009;49:553-567.)

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Section: Resultsmentioning

confidence: 99%

Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing

et al. 2008

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“…TMAO is a naturally occurring osmolyte that counteracts the destabilizing interaction of proteins with urea in different marine elasmobranchs (30). It was also shown to increase the melting temperature of proteins in vitro (31,32) and is effective in correcting temperature-sensitive protein folding abnormalities in vivo (33). In contrast to various polyols that stabilize collagens and collagenous peptides (23,29,34), TMAO has an excellent far-UV transparency.…”

Section: Resultsmentioning

confidence: 99%

Destabilization of osteogenesis imperfecta collagen-like model peptides correlates with the identity of the residue replacing glycine

Beck

Chan

Shenoy

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

179

198

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Mutations resulting in replacement of one obligate Gly residue within the repeating (Gly-Xaa-Yaa) n triplet pattern of the collagen type I triple helix are the major cause of osteogenesis imperfecta (OI). Phenotypes of OI involve fragile bones and range from mild to perinatal lethal. In this study, host-guest triple-helical peptides of the form acetyl-(Gly-Pro-Hyp) 3-Zaa-Pro-Hyp-(Gly-Pro-Hyp)4-GlyGly-amide are used to isolate the influence of the residue replacing Gly on triple-helix stability, with Zaa ‫؍‬ Gly, Ala, Arg, Asp, Glu, Cys, Ser, or Val. Any substitution for Zaa ‫؍‬ Gly (melting temperature, Tm ‫؍‬ 45°C) results in a dramatic destabilization of the triple helix. For Ala and Ser, T m decreases to Ϸ10°C, and for the Arg-, Val-, Glu-, and Asp-containing peptides, Tm < 0°C. A Gly 3 Cys replacement results in T m < 0°C under reducing conditions but shows a broad transition (T m Ϸ 19°C) in an oxidizing environment. Addition of trimethylamine N-oxide increases T m by Ϸ5°C per 1 M trimethylamine N-oxide, resulting in stable triple-helix formation for all peptides and allowing comparison of relative stabilities. The order of disruption of different Gly replacements in these peptides can be represented as Ala < Ser < CPOred < Arg < Val < Glu < Asp. The rank of destabilization of substitutions for Gly in these Gly-ProHyp-rich homotrimeric peptides shows a significant correlation with the severity of natural OI mutations in the ␣1 chain of type I collagen.

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“…The most important question is whether the folding of a protein can be influenced specifically, with the goals either of curing a disease or of preventing virus assembly. Chemical chaperones and ligand-induced transport opens options for designing specific drugs to control protein (mis)folding or transport 37,38 . Likewise, tissue-specific chaperones might be therapeutic targets and might provide important tools in biotechnology.…”

Section: Perspectivesmentioning

confidence: 99%