David Pugh

Tetratricopeptide Repeat Motif-mediated Hsc70-mSTI1 Interaction MOLECULAR CHARACTERIZATION OF THE CRITICAL CONTACTS FOR SUCCESSFUL BINDING AND SPECIFICITY

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human Hsp70/Hsp90-organizing protein (Hop). Guided by Hop structural data and sequence alignment analyses, we have used site-directed mutagenesis, co-precipitation assays, circular dichroism spectroscopy, steady-state fluorescence, and surface plasmon resonance spectroscopy to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal tetratricopeptide repeat domain (TPR1) of mSTI1 to bind to heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. We have shown that substitutions in the first TPR motif of Lys8 or Asn12did not affect binding of mSTI1 to Hsc70, whereas double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn43 lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys8 or Asn12 reduced but did not abrogate binding. These results suggest that mSTI1-Hsc70 interaction requires a network of interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70 but also outside the TPR domain. We propose that the electrostatic interactions in the first TPR motif made by Lys8 or Asn12 define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, we substituted residues on TPR1 potentially involved in hydrophobic contacts with Hsc70. The modified protein had reduced binding to Hsc70 but now showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on TPR2A. Our results suggest that binding of Hsc70 to TPR1 is more specific than binding of Hsp90 to TPR2A with serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in vivo.

DWNN, a novel ubiquitin-like domain, implicates RBBP6 in mRNA processing and ubiquitin-like pathways

BackgroundRBBP6 is a 250 kDa splicing-associated protein that has been identified as an E3 ligase due to the presence of a RING finger domain. In humans and mice it interacts with both p53 and Rb, and plays a role in the induction of apoptosis and regulation of the cell cycle. RBBP6 has recently been shown to be highly up-regulated in oesophageal cancer, and to be a promising target for immunotherapy against the disease.ResultsWe show here using heteronuclear NMR that the N-terminal 81 amino acids of RBBP6 constitute a novel ubiquitin-like domain, which we have called the DWNN domain. The domain lacks conserved equivalents of K48 and K63, although the equivalents of K6 and K29 are highly, although not absolutely, conserved. The di-glycine motif that is characteristic of proteins involved in ubiquitination is found in the human and mouse form of the domain, although it is not present in all organisms. It forms part of a three-domain form of RBBP6 containing the DWNN domain, a zinc knuckle and a RING finger domain, which is found in all eukaryotic genomes so far examined, in the majority of cases at single copy number. The domain is also independently expressed in vertebrates as a single domain protein.ConclusionDWNN is a novel ubiquitin-like domain found only at the N-terminus of the RBBP6 family of splicing-associated proteins. The ubiquitin-like structure of the domain greatly increases the likelihood that RBBP6 functions through some form of ubiquitin-like modification. Furthermore, the fact that the DWNN domain is independently expressed in higher vertebrates leads us to propose that the domain may itself function as a novel ubiquitin-like modifier of other proteins.

RBBP6 Interacts with Multifunctional Protein YB-1 through Its RING Finger Domain, Leading to Ubiquitination and Proteosomal Degradation of YB-1

RBBP6 (retinoblastoma binding protein 6) is a 250-kDa multifunctional protein that interacts with both p53 and pRb and has been implicated in mRNA processing. It has also been identified as a putative E3 ubiquitin ligase due to the presence of a RING finger domain, although no substrate has been identified up to now. Using the RING finger domain as bait in a yeast two-hybrid screen, we identified YB-1 (Y-box binding protein 1) as a binding partner of RBBP6, localising the interaction to the last 62 residues of YB-1. We showed, furthermore, that both full-length RBBP6 and the isolated RING finger domain were able to ubiquitinate YB-1, resulting in its degradation in the proteosome. As a result, RBBP6 was able to suppress the levels of YB-1 in vivo and to reduce its transactivational ability. In the light of the important role that YB-1 appears to play in tumourigenesis, our results suggest that RBBP6 may be a relevant target for therapeutic drugs aimed at modifying the activity of YB-1.

The RBBP6/ZBTB38/MCM10 Axis Regulates DNA Replication and Common Fragile Site Stability

Faithful DNA replication is essential for the maintenance of genome integrity. Incomplete genome replication leads to DNA breaks and chromosomal rearrangements, which are causal factors in cancer and other human diseases. Despite their importance, the molecular mechanisms that control human genome stability are incompletely understood. Here, we report a pathway that is required for human genome replication and stability. This pathway has three components: an E3 ubiquitin ligase, a transcriptional repressor, and a replication protein. The E3 ubiquitin ligase RBBP6 ubiquitinates and destabilizes the transcriptional repressor ZBTB38. This repressor negatively regulates transcription and levels of the MCM10 replication factor on chromatin. Cells lacking RBBP6 experience reduced replication fork progression and increased damage at common fragile sites due to ZBTB38 accumulation and MCM10 downregulation. Our results uncover a pathway that ensures genome-wide DNA replication and chromosomal stability.

Solution Structure of RING Finger-like Domain of Retinoblastoma-binding Protein-6 (RBBP6) Suggests It Functions as a U-box

Background: U-box-containing proteins cooperate with chaperones in ubiquitinating irreversibly unfolded proteins. Results: Retinoblastoma binding protein-6 (RBBP6) contains a zinc-binding U-box-like domain and interacts directly with chaperones. Conclusion: RBBP6 may play a role in protein quality control. Significance: U-boxes should be classified in terms of their interaction with chaperones and not their zinc binding properties. Retinoblastoma-binding protein-6 (RBBP6) plays a facilitating role, through its RING finger-like domain, in the ubiquitination of p53 by Hdm2 that is suggestive of E4-like activity. Although the presence of eight conserved cysteine residues makes it highly probable that the RING finger-like domain coordinates two zinc ions, analysis of the primary sequence suggests an alternative classification as a member of the U-box family, the members of which do not bind zinc ions. We show here that despite binding two zinc ions, the domain adopts a homodimeric structure highly similar to those of a number of U-boxes. Zinc ions could be replaced by cadmium ions without significantly disrupting the structure or the stability of the domain, although the rate of substitution was an order of magnitude slower than any previous measurement, suggesting that the structure is particularly stable, a conclusion supported by the high thermal stability of the domain. A hallmark of U-box-containing proteins is their association with chaperones, with which they cooperate in eliminating irretrievably unfolded proteins by tagging them for degradation by the proteasome. Using a yeast two-hybrid screen, we show that RBBP6 interacts with chaperones Hsp70 and Hsp40 through its N-terminal ubiquitin-like domain. Taken together with the structural similarities to U-box-containing proteins, our data suggest that RBBP6 plays a role in chaperone-mediated ubiquitination and possibly in protein quality control.

Abstract 1085: RbBP6 isoform 3/DWNN is essential for arsenic trioxide-induced G2/M arrest in cancer cell lines

Retinoblastoma binding protein 6 (RbBP6) is a gene that encodes RbBP6 protein products (isoforms 1,2 and 3) that has previously been documented to bind retinoblastoma protein (pRb) and p53, which are key components of cell cycle regulation and localises on chromosome 16p12.2 in humans. These isoforms are derived from two major mRNA transcripts, a 1.1 kb (Isoform 3) and 6.1 kb (isoform 1and 2). These two tumour suppressors are important to the understanding the function of the RbBP6. Arsenic trioxide has been used to treat APLS and has been shown to arrest cells at G1 and G2. In this study, the involvement of the DWNN in cell cycle arrest caused by As2O3 was investigated. RNA interference was employed to investigate the involvement of the DWNN in cell cycle regulation. Immunocytochemistry was employed to determine the localization of the DWNN-containing proteins in human cancer cells as well as non-cancerous cells. Propidium iodide cell cycle analysis was used to quantify cell cycle arrest induced by As2O3. Western blotting and real-time PCR were used to determine the expression of the DWNN in both control and treated cells. RNA interference was employed to investigate the involvement of the DWNN in cell cycle regulation. Immunocytochemistry was employed to determine the localization of the DWNN-containing proteins in human cancer cells as well as non-cancerous cells. Propidium iodide cell cycle analysis was utilised to quantify cell cycle arrest induced by As2O3. Western blotting and real-time PCR were used to determine the expression of the DWNN in both control and treated cells. DWNN knockdown increased cell proliferation and did not have an effect on apoptosis. Treatment of human cells with low concentrations of As2O3 induced G2 cell cycle arrest and resulted in induced expression of DWNN. This induced cell cycle arrest was also associated with deglycosylation of the DWNN domain. This finding is in line with the localization of the DWNN proteins in human cancer cells, which showed high expression of the DWNN proteins in G2/M cell cycle phase. Expression analysis showed that the up-regulation of the DWNN domain resulted in p53 up-regulation. These results strongly suggest that the DWNN may be either required for G2 cell cycle arrest or may be required for p53 stabilization during this phase or may be needed for signalling for progression to the next phase of cell cycle. DWNN involvement in cell cycle may be in a p53-dependent manner. Staurosporine which induces G1 cell cycle arrest did not result in DWNN up-regulation and that suggest that DWNN may not be required for G1 cell cycle arrest or regulation. The effect of DWNN in pRb phosphorylation and G1 cell cycle arrest still needs more attention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1085.

Abstract 1265: RbBP6 Isoform 3 (DWNN) is a p53 stabilizer in arsenic trioxide-induced apoptosis in human cancer cell lines

Domain With No Name (DWNN) has a ubiquitin-like fold and shares 22% similarity with ubiquitin. This suggests its significant importance in cell homeostasis and protein degradation in a human body. The presence of a ubiquitin-like fold suggests a role similar to ubiquitin and RbBP6 may be involved in ubiquitin ligase-like activities. Furthermore RbBP6 interacts with both p53 and pRb which are key components of cell cycle regulation. These two tumour suppressors are key to the understanding the function of the RbBP6. It has been found that upon arsenic trioxide-induced treatment RbBP6 isoform 3 (DWNN) is up-regulated. In this study the role of the DWNN on p53-dependent apoptosis was investigated. Over-expressions of RbBP6 isoforms was achieved by transfection of recombinant isoforms using Metafectene Easy. Apoptosis was induced using arsenic trioxide and staurosporine. Apoptosis was then measured using APOPercentage and flow cytometry. The expression of RbBP6 gene products and p53 was determined using both real-time PCR and western blotting. Up-regulation of RbBP6 was observed in As2O3-induced apoptosis in cancer cells. Over-expression of the RbBP6 isoform 1 intensified As2O3-induced apoptosis. MTT assay showed an IC50 of 40µM for As2O3. When this concentration was used to induce apoptosis in human cancer cells and this resulted in induced expression of RbBP6 isoform 1 not the isoform 3. Over-expression of RbBP6 isoform 3 though resulted in stabilization of p53 protein. The apoptosis that was observed was also accompanied by the expression of p53. This study suggests that the DWNN may not be directly involved in the prosecution of apoptosis but may be involved through p53 stabilization. This study suggests that the RbBP6 may be a p53 stabilizer in contrast to mdm2, which is a negative regulator of p53. It will be very important to also determine the relationship between RbBP6 and pRb in both cell cycle and apoptosis Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1265.