Identification of FAM96B as a novel prelamin A binding partner

https://doi.org/10.1016/j.bbrc.2013.08.099Get rights and content

Highlights

  • We screen the binding protein of prelamin A by yeast two-hybrid screen.

  • FAM96B colocalizes with prelamin A in HEK-293 cells.

  • FAM96B physically interacts with prelamin A.

Abstract

Prelamin A accumulation causes nuclear abnormalities, impairs nuclear functions, and eventually promotes cellular senescence. However, the underlying mechanism of how prelamin A promotes cellular senescence is still poorly understood. Here we carried out a yeast two-hybrid screen using a human skeletal muscle cDNA library to search for prelamin A binding partners, and identified FAM96B as a prelamin A binding partner. The interaction of FAM96B with prelamin A was confirmed by GST pull-down and co-immunoprecipitation experiments. Furthermore, co-localization experiments by fluorescent confocal microscopy revealed that FAM96B colocalized with prelamin A in HEK-293 cells. Taken together, our data demonstrated the physical interaction between FAM96B and prelamin A, which may provide some clues to the mechanisms of prelamin A in premature aging.

Introduction

Hutchinson–Gilford progeria syndrome (HGPS) is a severe childhood disease characterized by accelerated aging [1]. It is caused by mutations in LMNA, which encoding A-type lamins (predominantly lamin A and C) [2]. Lamin A is a component of the nuclear lamina that plays a critical role in the structural organization and function of the nucleus. Lamin A is first synthesized as a prelamin A precursor with a C-terminal CaaX motif and undergoes a series of posttranslational modifications including CaaX processing (farnesylation, aaX cleavage and carboxylmethylation), followed by endoproteolytic cleavage by Zmpste24 [3]. Loss of Zmpste24 activity arrests the processing of prelamin A at a stage similar to HGPS, although a unique truncated prelamin A (progerin) is accumulated in HGPS cells [4], [5], [6]. Recent studies have found that the similar molecular mechanism responsible for HGPS is active in healthy cells [7], [8]. Prelamin A accumulation is not observed in young healthy vessels but is prevalent in vascular smooth muscle cells from aged individuals, which accompanied with nuclear morphology defects. Therefore, the toxic accumulation of prelamin A might be a novel target to ameliorate the effects of age-induced cell dysfunction [8].

To better understand the deleterious effects of prelamin A accumulation, we performed a yeast two-hybrid screen on a human skeletal muscle cDNA library, using the full length of premanin A as a bait to search novel interacting factors. And the family with sequence similarity 96 member B (FAM96B, also known as MIP18) was hunted as a novel binding partner of prelamin A. We further validated their binding affinity using GST-pull down, co-immunoprecipitation and confocal co-localization experiments.

Section snippets

Yeast two-hybrid screen

A yeast two-hybrid screen was performed according to the Clontech Yeast Protocols Handbook (Clontech, Mountain View, CA). Briefly, cDNA encoding human prelamin A was inserted into the EcoR I-BamH I sites of the vector pGBKT7 containing the GAL-4 DNA binding domain (pGBKT7-prelamin A). The pGBKT7-prelamin A bait plasmid was transformed into yeast strain AH109, and then the transformed AH109 mated with yeast Y187 containing pACT2 with human skeletal muscle Matchmaker cDNA library. Positive clones

FAM96B was identified as a novel prelamin A-binding protein by yeast two-hybrid screen

To identify prelamin A-interacting proteins, we screened a human skeletal muscle cDNA library using the yeast two-hybrid system. With the full length prelamin A as bait, the positive clones, which specifically interacted with BD-prelamin A, but not BD-Null baits under both medium- and high-stringency situation, were sequenced and BLAST searched. FAM96B that had no auto-activation was identified as a potential partner, and then reintroduced into yeast cells to confirm the interaction with

Discussion

Growing evidences have suggested that prelamin A accumulation could induce DNA damage, leading to genomic instability, and premature senescence [5], [6], [8]. However, the underlying mechanism of how prelamin A promotes cellullar senescence is still poorly understood. Previous reports showed that prelamin A interacted with several proteins, including Narf [10], Emerin [11], SREBP1 [12] and SP1 [13]. In the present study, we screened prelamin A binding proteins by the yeast two-hybrid system,

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (81000143, 81370456, 30672205, 30871440, 81170327), the Natural Science Foundation of Guangdong Province (S2012010008219, S2011010002922, 9252402301000002), the Medical Scientific Research Foundation of Guangdong Province (B2009191, A2011431), the Science and Technology Planning Project for University Research Institutions and Medical and Health Organizations of Dongguan City (2011105102007), the Science &

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