The HCF repeat is an unusual proteolytic cleavage signal.

Abstract

The herpes simplex virus VPl6-associated protein HCF is a nuclear host-cell factor that exists as a family of polypeptides encoded by a single gene. The mature HCF polypeptides are amino-and carboxy-terminal fragments of a large -300-kD precursor protein that arise through cleavage at one or more centrally located sites. The sites of cleavage are the HCF repeats, highly conserved 26-amino-acid sequences repeated six times in the HCF precursor protein. The HCF repeat alone is sufficient to induce cleavage of a heterologous protein, and cleavage occurs at a defined site-PPCEITHET-within the HCF repeat. Alanine-scan mutagenesis was used to identify a large 18-amino-acid segment of the HCF repeat that is important to induce cleavage of a heterologous protein. Even though HCF is cleaved, the majority of amino-and carboxy-terminal cleavage products remain tightly, albeit noncovalently, associated. Modulation of this noncovalent association may provide a mechanism for regulating HCF activity. For example, the cleaved products of an alternative mRNA splicing variant of HCF do not remain associated.

Molecular cloning of Drosophila HCF reveals proteolytic processing and self‐association of the encoded protein

“…One function of the spacer region in HCF-1 is to specify proteolytic processing (Wilson et al, 1993b;Kristie et al, 1995;Wilson et al, 1995b;Vogel and Kristie, 2000). To determine if the dHCF protein is also processed, we transiently transfected Drosophila SL2 cells with an expression vector encoding full-length dHCF tagged at the N-terminus with the T7 epitope and at the C-terminus with a FLAG epitope (illustrated schematically in Fig.…”

“…In addition, HCF-1 contains its own activation domain and this is required for optimal transactivation by the VP16-induced complex ( The arrangement of functional domains in human HCF-1 is shown in Figure 1A. HCF-1 is translated as a 230-300-kDa precursor and processed into N-and C-terminal subunits through autocatalytic proteolytic cleavage at a series of six HCF PRO repeats located near the center of the precursor polypeptide (Wilson et al, 1993b;Kristie et al, 1995;Wilson et al, 1995b;Vogel and Kristie, 2000). The resulting subunits remain stably associated via two matched pairs of interaction domains: termed SAS1N-SAS1C and SAS2N-SAS2C .…”

“…The SAS2 self-association domains flank the HCF PRO repeat region and have been mapped with less precision. The majority of HCF-1 molecules in actively proliferating cells have undergone processing suggesting this is the active form of the protein (Wilson et al, 1993b;Kristie et al, 1995;Wilson et al, 1995b). …”

A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm

“…Briefly, they were constructed by PCR amplification with specific primers and subcloning into the pCGTHCF FL T7 expression vector (Wilson et al 1995), in which transcription is driven by the CMV enhancer/ promoter (Tanaka and Herr 1990;Cá ceres et al 1994) and the coding sequence begins with an amino-terminal epitope tag, MASMTGGQQMG. This epitope tag corresponds to the first 11 residues of the bacteriophage T7 gene 10 capsid protein and is recognized by the T7.tag monoclonal antibody (Novagen).…”

Autocatalytic proteolysis of the transcription factor-coactivator C1 (HCF): A potential role for proteolytic regulation of coactivator function

“…Previously, insertion of C1 processing sites in a donor protein (Oct-1) resulted in inefficient processing of the target in mammalian cells (41), suggesting that the C1 factor proteolytic activity may also function in trans. Therefore, this was investigated by using an E. coli coexpression assay as illustrated in Fig.…”