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Aberrant hetero-disulfide bond formation by the hypertriglyceridemia-associated p.Gly185Cys APOA5 variant (rs2075291).

ABSTRACT: Apolipoprotein A-V (apoA-V) is a low-abundance plasma protein that modulates triacylglycerol homeostasis. Gene transfer studies were undertaken in apoa5 (-/-) mice to define the mechanism underlying the correlation between the single-nucleotide polymorphism c.553G>T in APOA5 and hypertriglyceridemia.Adeno-associated virus (AAV) 2/8-mediated gene transfer of wild-type apoA-V induced a dramatic lowering of plasma triacylglycerol in apoa5 (-/-) mice, whereas AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T single-nucleotide polymorphism: rs2075291; p.Gly185Cys when numbering includes signal sequence) had a modest effect. Characterization studies revealed that plasma levels of wild-type and G162C apoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162C apoA-V indicated that, unlike wild-type apoA-V, >50% of G162C apoA-V was recovered in the lipoprotein-free fraction. Nonreducing SDS-PAGE immunoblot analysis provided evidence that G162C apoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked heterodimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for wild-type APOA5 and c.553G>T APOA5 revealed that G162C apoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1, and others.Substitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein-binding and triacylglycerol-modulation functions are compromised.

SUBMITTER: Sharma V

PROVIDER: S-EPMC4178935 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Project description:The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (?-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress. Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases. Yeast strains were constructed that produce and secrete (a) IP or (b) ?-amylase and were compared to yeast strains containing (c) an empty vector in both wild-type and HAC1 deletion backgrounds. These strains are named WN (WT with empty vector), WI (WT secreting IP), WA (WT secreting ?-amylase), dN (?hac1 with empty vector), dI (?hac1 secreting IP), and dA (?hac1 secreting ?-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.

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Aberrant hetero-disulfide bond formation by the hypertriglyceridemia-associated p.Gly185Cys APOA5 variant (rs2075291).

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