Biochemical and Biophysical Research Communications
Identification of FAM96B as a novel prelamin A binding partner
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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POLD1: Central mediator of DNA replication and repair, and implication in cancer and other pathologies
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FAM96B inhibits the senescence of dental pulp stem cells
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