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A regulator of secretory vesicle size, Kelch-like protein 12, facilitates the secretion of apolipoprotein B100 and very-low-density lipoproteins--brief report.

ABSTRACT: One of the major risk factors for atherosclerosis is the plasma level of low-density lipoprotein (LDL), which is a product of very-low-density lipoprotein (VLDL). Hepatic apolipoprotein B100 (apoB100) is the essential component that provides structural stability to VLDL particles. Newly translated apoB100 is partially lipidated in the endoplasmic reticulum (ER), forming nascent apoB100-VLDL particles. These particles are further modified to form fully mature VLDLs in the Golgi apparatus. Therefore, the transport of nascent VLDL from the ER to the Golgi represents a critical step during VLDL maturation and secretion and in regulating serum LDL cholesterol levels. Our previous studies showed that apoB100 exits the ER in coat complex II vesicles (COPII), but the cohort of related factors that control trafficking is poorly defined.Expression levels of Kelch-like protein 12 (KLHL12), an adaptor protein known to assist COPII-dependent transport of procollagen, were manipulated by using a KLHL12-specific small interfering RNA and a KLHL12 expression plasmid in the rat hepatoma cell line, McArdle RH7777. KLHL12 knockdown decreased the secreted and intracellular pools of apoB100, an effect that was attenuated in the presence of an autophagy inhibitor. KLHL12 knockdown also significantly reduced secretion of the most lipidated apoB100-VLDL species and led to the accumulation of apoB100 in the ER. Consistent with these data, KLHL12 overexpression increased apoB100 recovery and apoB100-VLDL secretion. Images obtained from confocal microscopy revealed colocalization of apoB100 and KLHL12, further supporting a direct link between KLHL12 function and VLDL trafficking from the ER.KLHL12 plays a critical role in facilitating the ER exit and secretion of apoB100-VLDL particles, suggesting that KLHL12 modulation would influence plasma lipid levels.

SUBMITTER: Butkinaree C

PROVIDER: S-EPMC3919549 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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A regulator of secretory vesicle size, Kelch-like protein 12, facilitates the secretion of apolipoprotein B100 and very-low-density lipoproteins--brief report.

Butkinaree Chutikarn C Guo Liang L Ramkhelawon Bhama B Wanschel Amarylis A Brodsky Jeffrey L JL Moore Kathryn J KJ Fisher Edward A EA

Arteriosclerosis, thrombosis, and vascular biology 20131212 2

<h4>Objective</h4>One of the major risk factors for atherosclerosis is the plasma level of low-density lipoprotein (LDL), which is a product of very-low-density lipoprotein (VLDL). Hepatic apolipoprotein B100 (apoB100) is the essential component that provides structural stability to VLDL particles. Newly translated apoB100 is partially lipidated in the endoplasmic reticulum (ER), forming nascent apoB100-VLDL particles. These particles are further modified to form fully mature VLDLs in the Golgi ...[more]

PMID: 24334870

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Project description:RATIONALE:Genome-wide association studies identified single-nucleotide polymorphisms near the SORT1 locus strongly associated with decreased plasma LDL-C (low-density lipoprotein cholesterol) levels and protection from atherosclerotic cardiovascular disease and myocardial infarction. The minor allele of the causal SORT1 single-nucleotide polymorphism locus creates a putative C/EBP? (CCAAT/enhancer-binding protein ?)-binding site in the SORT1 promoter, thereby increasing in homozygotes sortilin expression by 12-fold in liver, which is rich in this transcription factor. Our previous studies in mice have showed reductions in plasma LDL-C and its principal protein component, apoB (apolipoprotein B) with increased SORT1 expression, and in vitro studies suggested that sortilin promoted the presecretory lysosomal degradation of apoB associated with the LDL precursor, VLDL (very-low-density lipoprotein). OBJECTIVE:To determine directly that SORT1 overexpression results in apoB degradation and to identify the mechanisms by which this reduces apoB and VLDL secretion by the liver, thereby contributing to understanding the clinical phenotype of lower LDL-C levels. METHODS AND RESULTS:Pulse-chase studies directly established that SORT1 overexpression results in apoB degradation. As noted above, previous work implicated a role for lysosomes in this degradation. Through in vitro and in vivo studies, we now demonstrate that the sortilin-mediated route of apoB to lysosomes is unconventional and intersects with autophagy. Increased expression of sortilin diverts more apoB away from secretion, with both proteins trafficking to the endosomal compartment in vesicles that fuse with autophagosomes to form amphisomes. The amphisomes then merge with lysosomes. Furthermore, we show that sortilin itself is a regulator of autophagy and that its activity is scaled to the level of apoB synthesis. CONCLUSIONS:These results strongly suggest that an unconventional lysosomal targeting process dependent on autophagy degrades apoB that was diverted from the secretory pathway by sortilin and provides a mechanism contributing to the reduced LDL-C found in individuals with SORT1 overexpression.

Project description:Glucosamine impairs hepatic apolipoprotein B100 (apoB100) production by inducing endoplasmic reticulum (ER) stress and enhancing cotranslational and posttranslational apoB100 degradation (Qiu, W., R. K. Avramoglu, A. C. Rutledge, J. Tsai, and K. Adeli. Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins. J. Lipid Res. 2006. 47: 1749-1761). Here, we report that glucosamine also regulates apoB100 protein synthesis via ER-stress-induced PERK activation. Short-term (4 h) glucosamine treatment of HepG2 cells reduced both cellular (by 62%) and secreted apoB100 (by 43%) without altering apoB100 mRNA. Treatment with proteasomal inhibitors only partially prevented the suppressive effects of glucosamine, suggesting that mechanisms other than proteasomal degradation may also be involved. Glucosamine-induced ER stress was associated with a significantly reduced apoB100 synthesis with no significant change in posttranslational decay rates, suggesting that glucosamine exerted its effect early during apoB biosynthesis. The role of PERK and its substrate, alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), in the suppressive effects of glucosamine on apoB synthesis was then investigated. Coexpression of apoB15 (normally resistant to intracellular degradation) with wild-type double stranded (ds) RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) in COS-7 cells resulted in a dramatic reduction in the levels of newly synthesized apoB15. Interestingly, cotransfection with apoB15 and a kinase inactive PERK mutant (K618A) increased apoB15 expression. In addition, short-term glucosamine treatment stimulated an increase in phosphorylation of PERK and eIF2alpha. Taken together, these data suggest that in addition to the induction of ER-associated degradation and other degradative pathways, ER stress is associated with suppression of apoB synthesis via a PERK-dependent mechanism.

Dataset Information

A regulator of secretory vesicle size, Kelch-like protein 12, facilitates the secretion of apolipoprotein B100 and very-low-density lipoproteins--brief report.

Publications

A regulator of secretory vesicle size, Kelch-like protein 12, facilitates the secretion of apolipoprotein B100 and very-low-density lipoproteins--brief report.

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