NMR and mass spectrometry studies of putative interactions of cell cycle proteins pRb and CDK6 with cell differentiation proteins MyoD and ID-2.

Cell growth and differentiation require precise coordination of cell cycle and differentiation proteins. This can be achieved by direct interactions between proteins, by indirect interaction in multiprotein complexes, or by modulation of gene expression levels of partner proteins. Contradictory data abound in the literature regarding the binding between some central cell cycle proteins, pRb, and CDK6, with myogenic differentiation promoting, MyoD, and inhibiting, Id-2, factors. We have tested these interactions using pure proteins and in vitro biophysical and biochemical methods, which included mass spectrometry, nuclear magnetic resonance (NMR), the affinity chromatography pull-down assays, and gel filtration chromatography. Using this multimethod approach, we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, CDK6 and p19, and MyoD and DNA. Using the same methods, we could unambiguously show that there is no direct protein-protein interaction in vitro between the small pocket domain of pRb and the bHLH domain of MyoD, the small pocket domain of pRb and Id-2, and CDK6 and a 15-amino-acid peptide from the C-terminal domain of MyoD. Indirect interactions, through additional binding partners in multiprotein complexes or modulation of gene expression levels of these proteins, are therefore their probable mode of action.

Chelating agents stabilize the monomeric state of the zinc binding human papillomavirus 16 E6 oncoprotein.

The E6 protein of human papillomavirus 16 is known to be difficult and, when overexpressed, insoluble and agglomerated. It has two putative zinc ion binding sites crucial for its function. No metallochaperone has yet been found to deliver zinc ions to the E6 protein. Here, we report that a specific chelating agent, which we think functionally mimics a metallochaperone, stabilized the soluble monomeric form of E6 and inhibited multimerization in vitro. This effect seemed to depend on the chelating strength of the agent. While strong chelating agents precipitated the E6 protein and weak chelating agents did not favor the monomeric form of E6, chelating agents of intermediate strength [L-penicillamine and ethylene glycol bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA)] effectively support the formation of a monomer. We did not observe formation of a dimer or defined oligomers. Degradation assays imply that the monomer is the biologically active form of the protein. Since EGTA favors the formation of monomeric over agglomerated E6 protein, we propose that chelating agents of appropriate strength could assist zinc delivery to recombinant metalloproteins in vitro and may even destabilize existing agglomerates.

Interaction with CBP/p300 enables the bovine papillomavirus type 1 E6 oncoprotein to downregulate CBP/p300-mediated transactivation by p53.

The E6 oncoprotein of bovine papillomavirus type 1 (BPV-1) can transform cells independently of p53 degradation. The precise mechanisms underlying this transformation are not yet completely understood. Here it is shown that BPV-1 E6 interacts with CBP/p300 in the same way as described for the E6 proteins of oncogenic human papillomaviruses. This interaction results in an inhibition of the transcriptional coactivator function of CBP/p300 required by p53 and probably by other transcription factors. The comparison of the CBP/p300-binding properties of BPV-1 E6 mutants previously characterized in transcription and transformation studies suggests (i) that the E6-CBP/p300 interaction may be necessary, but not sufficient, for cell transformation, and (ii) that the transcriptional activator function, inherent to the E6 protein, is not derived from forming a complex with CBP/p300.

The human papillomavirus type 11 and 16 E6 proteins modulate the cell-cycle regulator and transcription cofactor TRIP-Br1.

The genital human papillomaviruses (HPVs) are a taxonomic group including HPV types that preferentially cause genital and laryngeal warts ("low-risk types"), such as HPV-6 and HPV-11, or cancer of the cervix and its precursor lesions ("high-risk types"), such as HPV-16. The transforming processes induced by these viruses depend on the proteins E5, E6, and E7. Among these oncoproteins, the E6 protein stands out because it supports a particularly large number of functions and interactions with cellular proteins, some of which are specific for the carcinogenic HPVs, while others are shared among low- and high-risk HPVs. Here we report yeast two-hybrid screens with HPV-6 and -11 E6 proteins that identified TRIP-Br1 as a novel cellular target. TRIP-Br1 was recently detected by two research groups, which described two separate functions, namely that of a transcriptional integrator of the E2F1/DP1/RB cell-cycle regulatory pathway (and then named TRIP-Br1), and that of an antagonist of the cyclin-dependent kinase suppression of p16INK4a (and then named p34SEI-1). We observed that TRIP-Br1 interacts with low- and high-risk HPV E6 proteins in yeast, in vitro and in mammalian cell cultures. Transcription activation of a complex consisting of E2F1, DP1, and TRIP-Br1 was efficiently stimulated by both E6 proteins. TRIP-Br1 has an LLG E6 interaction motif, which contributed to the binding of E6 proteins. Apparently, E6 does not promote degradation of TRIP-Br1. Our observations imply that the cell-cycle promoting transcription factor E2F1/DP1 is dually targeted by HPV oncoproteins, namely (i) by interference of the E7 protein with repression by RB, and (ii) by the transcriptional cofactor function of the E6 protein. Our data reveal the natural context of the transcription activator function of E6, which has been predicted without knowledge of the E2F1/DP1/TRIP-Br/E6 complex by studying chimeric constructs, and add a function to the limited number of transforming properties shared by low- and high-risk HPVs.