Tissue factor pathway inhibitor-2 may interact with nuclear protein RASSF1C

Abstract

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region, three tandem Kunitz-type domains, and a positively charged carboxyterminal tail. Human TFPI-2 (hTFPI-2) inhibits a broad spectrum of serine proteinases (including trypsin, plasmin, plasma kallikrein, XIa, and chymotrypsin) almost exclusively via its first Kunitz-type domain, and potentially plays an important role in the regulation of extracellular matrix digestion and remodeling [1]. Reduced TFPI-2 synthesis has been related to numerous pathophysiological processes such as inflammation, angiogenesis, atherosclerosis [2,3], retinal degeneration, and tumor growth/metastasis [4–6]. It has been suggested that TFPI-2 is a tumor suppressor gene in some cancers [7,8]. However, the specific physiological functions of hTFPI-2 in humans are unclear, particularly its interactions with other proteins. To better understand the physiological function of hTFPI-2, we used yeast two-hybrid system screening and bioinformatics analysis to identify its interacting proteins and confirm its interactions with nuclear protein RASSF1C using confocal microscopy and co-immunoprecipitation. We used pre-hFB cDNA library from a number of libraries that are compatible with the system by Clontech company’s guide. By using the Matchmaker 3 yeast two-hybrid system, we screened 95 coding genes that may interact with TFPI-2. The entire human TFPI-2 open reading frame was ligated in-frame to the carboxy-terminus of the GAL4 DNA-binding domain supplied in plasmid pGBKT7. The resulting construct, pGBKT7-hTFPI-2, provided the ‘bait’ for the interaction trap screen. The bait-plasmid pGBKT7-hTFPI2 was transformed into the activated AH109 yeast and cultured clones were picked while 2– 3 mm in diameter. pGBKT7-hTFPI-2/AH109 (the ‘bait’) was inoculated into medium containing SD/-Trp mix. Yeast mating was performed with strain AH109 harboring bait plasmid and Y187 pre-transformed with a human fetal brain cDNA library constructed with a pGADT7-Rec vector. The mating mixture was plated onto SD/-Ade/-His/-Leu/-Trp (QDO) with the chromogenic substrate X-a-Gal allowing for color selection indicating protein –protein interaction. Within 8–21 days, blue colonies were isolated and the plasmid DNA was extracted by using a yeast plasmid DNA isolation kit. The AD and LD insert screening primer set (AD5: 5 0 -CTATTCGATGAT GAAGATACCCCACCAAACCC-3 0 and AD3: 5 0 -GTGAA CTTGCGGGGTTTTTCAGTATCTACGAT-3 0 ) was used to amplify the library inserted sequences by polymerase chain reaction (PCR). PCR products were identified on a 1% agarose gel and positive bands were extracted and sequenced. Sequencing results were blasted in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). DNA sequencing was conducted by Shanghai Invitrogen Biotechnology Co. (Shanghai, China). The DNA sequence of the cDNA inserts was determined. Blast searching of all sequences revealed 125 different