Species selectivity of mixed-lineage leukemia/trithorax and HCF proteolytic maturation pathways

Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.     

HCF-1 amino- and carboxy-terminal subunit association through two separate sets of interaction modules: involvement of fibronectin type 3 repeats

When herpes simplex virus infects permissive cells, the viral regulatory protein VP16 forms a specific complex with HCF-1, a preexisting nuclear protein involved in cell proliferation. The majority of HCF-1 in the cell is a complex of associated amino (HCF-1(N))- and carboxy (HCF-1(C))-terminal subunits that result from an unusual proteolytic processing of a large precursor polypeptide. Here, we have characterized the structure and function of sequences required for HCF-1(N) and HCF-1(C) subunit association. HCF-1 contains two matched pairs of self-association sequences called SAS1 and SAS2. One of these matched association sequences, SAS1, consists of a short 43-amino-acid region of the HCF-1(N) subunit, which associates with a carboxy-terminal region of the HCF-1(C) subunit that is composed of a tandem pair of fibronectin type 3 repeats, a structural motif known to promote protein-protein interactions. Unexpectedly, the related protein HCF-2, which is not proteolyzed, also contains a functional SAS1 association element, suggesting that this element does not function solely to maintain HCF-1(N) and HCF-1(C) subunit association. HCF-1(N) subunits do not possess a nuclear localization signal. We show that, owing to a carboxy-terminal HCF-1 nuclear localization signal, HCF-1(C) subunits can recruit HCF-1(N) subunits to the nucleus.     

O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1

The human epigenetic cell-cycle regulator HCF-1 undergoes an unusual proteolytic maturation process resulting in stably associated HCF-1(N) and HCF-1(C) subunits that regulate different aspects of the cell cycle. Proteolysis occurs at six centrally located HCF-1(PRO)-repeat sequences and is important for activation of HCF-1(C)-subunit functions in M phase progression. We show here that the HCF-1(PRO) repeat is recognized by O-linked β-N-acetylglucosamine transferase (OGT), which both O-GlcNAcylates the HCF-1(N) subunit and directly cleaves the HCF-1(PRO) repeat. Replacement of the HCF-1(PRO) repeats by a heterologous proteolytic cleavage signal promotes HCF-1 proteolysis but fails to activate HCF-1(C)-subunit M phase functions. These results reveal an unexpected role of OGT in HCF-1 proteolytic maturation and an unforeseen nexus between OGT-directed O-GlcNAcylation and proteolytic maturation in HCF-1 cell-cycle regulation.     

Analysis of functional domains of the host cell factor involved in VP16 complex formation.

We present biochemical analyses of the regions of the host cell factor (HCF) involved in VP16 complex formation and in the association between the N- and C-terminal domains of HCF itself. We show that the kelch repeat region of HCF (residues 1-380) is sufficient for VP16 complex formation, but that residues C-terminal to the repeats (positions 381-450) interfere with this activity. However, these latter residues are required for the interaction between the N- and C-terminal regions of HCF. The extreme C-terminal region of HCF, corresponding to an area of strong conservation with a Caenorhabditis elegans homologue, is sufficient for interaction with the N-terminal region. These results are discussed with respect to possible differences in the roles of HCF in VP16 activity versus its normal cellular function.     

Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins

A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.     

Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain.

Mammalian DNA (cytosine-5) methyltransferase contains a C-terminal domain that is closely related to bacterial cytosine-5 restriction methyltransferase. This methyltransferase domain is linked to a large N-terminal domain. It is shown here that the N-terminal domain contains a Zn binding site and that the N- and C-terminal domains can be separated by cleavage with trypsin or Staphylococcus aureus protease V8; the protease V8 cleavage site was determined by Edman degradation to lie 10 residues C-terminal of the run of alternating lysyl and glycyl residues which joins the two domains and six residues N-terminal of the first sequence motif conserved between the mammalian and bacterial cytosine methyltransferases. While the intact enzyme had little activity on unmethylated DNA substrates, cleavage between the domains caused a large stimulation of the initial velocity of methylation of unmethylated DNA without substantial change in the rate of methylation of hemimethylated DNA. These findings indicate that the N-terminal domain of DNA methyltransferase ensures the clonal propagation of methylation patterns through inhibition of the de novo activity of the C-terminal domain. Mammalian DNA methyltransferase is likely to have arisen via fusion of a prokaryotic-like restriction methyltransferase and an unrelated DNA binding protein. Stimulation of the de novo activity of DNA methyltransferase by proteolytic cleavage in vivo may contribute to the process of ectopic methylation observed in the DNA of aging animals, tumors and in lines of cultured cells.