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Biomedical subjects

Martin Scheffner

Publications and source records attributed to Martin Scheffner.

17 recordsLinked to original sources
PubMed2006

The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins.

The E6 oncoprotein of human papillomaviruses associated with cervical cancer targets the tumor suppressor p53 and several other cellular proteins including the human homologs of Dlg and Scribble for degradation via the ubiquitin-proteasome system. Similar to p53 degradation, E6-induced degradation of Scribble is mediated by the ubiquitin ligase E6-AP. In contrast, degradation of Dlg in vitro and within cells has been reported to be independent of E6-AP, suggesting that the E6 oncoprotein has the ability to interact with ubiquitin ligases other than E6-AP. Furthermore, the ability of the E6 oncoprotein to interact with these yet unidentified ubiquitin ligases may be shared by the E6 protein of so-called low risk human papillomaviruses that are not associated with cervical cancer. In this study, we used the RNA interference technology and mouse embryo fibroblasts derived from E6-AP-deficient mice to obtain information about the identity of the ubiquitin ligase(s) involved in E6-mediated degradation of Dlg. We report that, within cells, E6-mediated degradation of Dlg depends on the presence of functional E6-AP and provide evidence that the E6 protein of low risk human papillomaviruses functionally interacts with E6-AP. Based on these data, we propose that, in general, the proteolytic properties of human papillomavirus E6 proteins are mediated by interaction with E6-AP.

Animals↗
PubMed2006

Regulation of p27 degradation and S-phase progression by Ro52 RING finger protein.

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27 provides a powerful route for enforcing normal progression through the mammalian cell cycle. According to a current model, the ubiquitination of p27 during S-phase progression is mediated by SCF(Skp2) E3 ligase that captures Thr187-phosphorylated p27 by means of the F-box protein Skp2, which in turn couples the bound substrate via Skp1 to a catalytic core complex composed of Cul1 and the Rbx/Roc RING finger protein. Here we identify Skp2 as a component of an Skp1-cullin-F-box complex that is based on a Cul1-Ro52 RING finger B-box coiled-coil motif family protein catalytic core. Ro52-containing complexes display E3 ligase activity and promote the ubiquitination of Thr187-phosphorylated p27 in a RING-dependent manner in vitro. The knockdown of Ro52 expression in human cells with small interfering RNAs causes the accumulation of p27 and the failure of cells to enter S phase. Importantly, these effects are abrogated by the simultaneous removal of p27. Taken together, these data suggest a key role for Ro52 RING finger protein in the regulation of p27 degradation and S-phase progression in mammalian cells and provide evidence for the existence of a Cul1-based catalytic core that utilizes Ro52 RING protein to promote ubiquitination.

Amino Acid Motifs↗
PubMed2005

The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation.

The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.

Amino Acid Sequence↗
PubMed2005

Oncolytic adenovirus expressing a p53 variant resistant to degradation by HPV E6 protein exhibits potent and selective replication in cervical cancer.

Rationale in the development of novel treatment strategies for HPV-associated cancers is targeting on the basis of the presence of HPV in (pre)malignant cells. Here, we designed a new conditionally replicating adenovirus (CRAd) for selective and effective oncolytic replication in HPV-containing cells. As the backbone, we used the CRAd AdCB016, which replicates selectively in cells expressing HPV E6 and E7 proteins. To enhance its oncolytic potency, we armed AdCB016 with p53 variant mp53(268N), which is resistant to HPV E6-mediated degradation. The new CRAd AdCB016-mp53(268N) was analyzed for its lytic replication properties in cervical carcinoma cell lines, HPV-immortalized keratinocyte cell lines representing dysplastic cells, and primary human keratinocytes. AdCB016-mp53(268N) exhibited 10- to 1000-fold greater efficacy than AdCB016 on high-risk HPV-positive cervical carcinoma cells and HPV-immortalized keratinocytes. Importantly, infection with AdCB016-mp53(268N) did not affect primary nonmalignant human keratinocytes. This favorable efficacy and safety profile was confirmed in organotypic raft cultures. Our findings suggest that AdCB016-mp53(268N) is a promising new agent for treatment of HPV-associated human cancers.

Adenoviridae↗
PubMed2005

The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation.

The cellular level of the tumor suppressor p53 is tightly regulated through induced degradation via the ubiquitin/proteasome system. The ubiquitin ligase Mdm2 plays a pivotal role in stimulating p53 turnover. However, recently additional ubiquitin ligases have been identified that participate in the degradation of the tumor suppressor. Apparently, multiple degradation pathways are employed to ensure proper destruction of p53. Here we show that the chaperone-associated ubiquitin ligase CHIP is able to induce the proteasomal degradation of p53. CHIP-induced degradation was observed for mutant p53, which was previously shown to associate with the chaperones Hsc70 and Hsp90, and for the wild-type form of the tumor suppressor. Our data reveal that mutant and wild-type p53 transiently associate with molecular chaperones and can be diverted onto a degradation pathway through this association.

Cell Line↗
PubMed2005

Growth suppression induced by downregulation of E6-AP expression in human papillomavirus-positive cancer cell lines depends on p53.

The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells. These data indicate that the anti-growth-suppressive properties of E6-AP in HPV-positive cells depend on its ability to induce p53 degradation.

Down-Regulation↗
PubMed2004

Role of the histone acetyl transferase Tip60 in the p53 pathway.

The histone acetyl transferase Tip60 (HTATIP) shares many properties with the tumor suppressor p53 (TP53). Both proteins are involved in the cellular response to DNA damage, are subjected to proteasomal digestion following Mdm2-mediated ubiquitination, and accumulate after UV irradiation. We found here that knock-down of Tip60 affects the p53-dependent response following actinomycin D treatment, most likely because it inhibits p21 (CDKN1A) accumulation. Moreover, Tip60 is required for p53 to activate the endogenous p21 promoter, suggesting that it functions as a p53 co-activator. However, we also found that knock-down of Tip60 increases the turnover rate of p53 under normal growth conditions. Tip60 interferes with Mdm2-mediated degradation of p53, probably because it affects its subcellular localization. Taken together, our results suggest that Tip60 plays a double role in the p53 pathway: under normal growth conditions, Tip60 contributes to maintain a basal pool of p53 by interfering with its degradation; following DNA damage, Tip60 functions as p53 co-activator. That these two distinct roles are linked during the p53-dependent response is an attractive hypothesis.

Acetyltransferases↗
PubMed2003

The ubiquitin-protein ligase activity of Hdm2 is inhibited by nucleic acids.

The proto-oncoprotein Hdm2 is a member of the RING finger-type family of ubiquitin-protein ligases E3. The RING finger domain is assumed to mediate the specific interaction of an E3 with its cognate ubiquitin-conjugating enzyme E2, which catalyzes the covalent attachment of ubiquitin to substrate proteins. In addition, the RING finger domain of Hdm2 is involved in Hdm2 homooligomer formation and has the capacity to bind to RNA in a sequence-specific manner. Here we report that interaction with nucleic acids interferes with both Hdm2/Hdm2 complex formation and auto-ubiquitination of Hdm2 in vitro. Furthermore, although binding of Hdm2 to the tumor suppressor p53 is not inhibited by nucleic acids, Hdm2-mediated ubiquitination of p53 is significantly decreased. Taken together, these results provide the first example of an E3 whose activity can be regulated by direct interaction with nucleic acids.

Base Sequence↗
PubMed2003

HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53.

The RING finger proteins HdmX and Hdm2 share significant structural and functional similarity. Hdm2 is a member of the RING finger family of ubiquitin-protein ligases E3 and targets the tumor suppressor protein p53 for degradation. Although HdmX also binds to p53, HdmX does not induce p53 degradation. Moreover, HdmX has been reported to interfere with p53 degradation in overexpression experiments. To obtain insight into the mechanism by which HdmX interferes with p53 degradation, we studied the effect of HdmX on the E3 activity of Hdm2 in vitro. Surprisingly, this revealed that HdmX stimulates Hdm2-mediated ubiquitination of p53 and that HdmX facilitates ubiquitination of Hdm2 and vice versa. In addition, down-regulation of HdmX expression within cells results in the accumulation of both p53 and Hdm2. Because HdmX alone does not have appreciable E3 activity, these data indicate that HdmX acts as a stimulator, rather than as an inhibitor, of the E3 activity of Hdm2 and that, at least under certain conditions, HdmX is actively involved in the degradation of both p53 and Hdm2.

Amino Acid Sequence↗
PubMed2003

siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells.

The targeted inhibition of antiapoptotic factors in tumour cells may provide a rational approach towards the development of novel anticancer therapies. Using human papillomavirus (HPV)-transformed cells as a model system, we investigated if RNA interference (RNAi)-mediated gene silencing can be employed in order to overcome the apoptosis resistance of cancer cells. We found that both vector-borne and synthetic small interfering (si)RNAs, specifically directed against the antiapoptotic HPV E6 oncogene, restored dormant tumour suppressor pathways in HPV-positive cancer cells that are otherwise inactive in the presence of E6. This ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. These findings show that RNAi provides a powerful molecular strategy to inactivate intracellular E6 function efficiently. Moreover, they define E6 as a most promising therapeutic target to eliminate HPV-positive tumour cells specifically by RNAi. Thus, by sequence-specific targeting of antiapoptotic genes, siRNAs may be developed into novel therapeutics that can efficiently correct the apoptosis deficiency of cancer cells.

Apoptosis↗
PubMed2003

Human papillomavirus-induced carcinogenesis and the ubiquitin-proteasome system.

Certain types of human papillomaviruses have been etiologically associated with malignant lesions, most notably with cervical cancer. The major oncoproteins of these cancer-associated viruses are encoded by the viral E6 and E7 genes. Thorough characterization of these oncoproteins and their interaction with cellular proteins has shown that both E6 and E7 exploit the ubiquitin-proteasome system to degrade and, thus, to functionally inactivate negative cell-regulatory proteins including members of the p110(RB) family and p53. This act of piracy is assumed to contribute to both the efficient propagation of HPVs and HPV-induced carcinogenesis.

Cell Transformation, Neoplastic↗
PubMed2003

A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter.

The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat, which functions through binding to a short leader RNA, called transactivation responsive element (TAR). Recent studies suggest that Tat activates the HIV-1 long terminal repeat (LTR), mainly by adapting co-activator complexes, such as p300, PCAF and the positive transcription elongation factor P-TEFb, to the promoter. Here, we show that the proto-oncoprotein Hdm2 interacts with Tat and mediates its ubiquitination in vitro and in vivo. In addition, Hdm2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination. Fusion of ubiquitin to Tat bypasses the requirement of Hdm2 for efficient transactivation, supporting the notion that ubiquitin has a non-proteolytic function in Tat-mediated transactivation.

Cell Line↗
PubMed2003

Involvement of the DNA repair protein hHR23 in p53 degradation.

The stability of the tumor suppressor protein p53 is regulated via the ubiquitin-proteasome-dependent proteolytic pathway. Like most substrates of this pathway, p53 is modified by the attachment of polyubiquitin chains prior to proteasome-mediated degradation. However, the mechanism(s) involved in the delivery of polyubiquitylated p53 molecules to the proteasome are currently unclear. Here, we show that the human DNA repair protein hHR23 binds to polyubiquitylated p53 via its carboxyl-terminal ubiquitin-associated (Uba) domain shielding p53 from deubiquitylation in vitro and in vivo. In addition, downregulation of hHR23 expression within cells by RNA interference results in accumulation of p53. Since the Ubl domain of hHR23 has been shown to interact with the 26S proteasome, we propose that hHR23 is intrinsically involved in the delivery of polyubiquitylated p53 molecules to the proteasome. In this model, the Uba domain of hHR23 binds to polyubiquitin chains formed on p53 and protects them from deubiquitylation, while the Ubl domain delivers the polyubiquitylated p53 molecules to the proteasome.

Animals↗
PubMed2002

Tip60 is targeted to proteasome-mediated degradation by Mdm2 and accumulates after UV irradiation.

Acetylation is a prominent post-translational modification of nucleosomal histone N-terminal tails, which regulates chromatin accessibility. Accordingly, histone acetyltransferases (HATs) play major roles in processes such as transcription. Here, we show that the HAT Tip60, which is involved in DNA repair and apoptosis following gamma irradiation, is subjected to proteasome-dependent proteolysis. Furthermore, we provide evidence that Mdm2, the ubiquitin ligase of the p53 tumour suppressor, interacts physically with Tip60 and induces its ubiquitylation and proteasome-dependent degradation. Moreover, a ubiquitin ligase-defective mutant of Mdm2 had no effect on Tip60 stability. Our results indicate that Mdm2 targets both p53 and Tip60, suggesting that these two proteins could be co-regulated with respect to protein stability. Consistent with this hypothesis, Tip60 levels increased significantly upon UV irradiation of Jurkat cells. Collectively, our results suggest that degradation of Tip60 could be part of the mechanism leading to cell transformation by Mdm2.

Acetyltransferases↗
PubMed2002

Ubiquitination-dependent cofactor exchange on LIM homeodomain transcription factors.

The interactions of distinct cofactor complexes with transcription factors are decisive determinants for the regulation of gene expression. Depending on the bound cofactor, transcription factors can have either repressing or transactivating activities. To allow a switch between these different states, regulated cofactor exchange has been proposed; however, little is known about the molecular mechanisms that are involved in this process. LIM homeodomain (LIM-HD) transcription factors associate with RLIM (RING finger LIM domain-binding protein) and with CLIM (cofactor of LIM-HD proteins; also known as NLI, Ldb and Chip) cofactors. The co-repressor RLIM inhibits the function of LIM-HD transcription factors, whereas interaction with CLIM proteins is important for the exertion of the biological activity conferred by LIM-HD transcription-factors. Here we identify RLIM as a ubiquitin protein ligase that is able to target CLIM cofactors for degradation through the 26S proteasome pathway. Furthermore, we demonstrate a ubiquitination-dependent association of RLIM with LIM-HD proteins in the presence of CLIM cofactors. Our data provide a mechanistic basis for cofactor exchange on DNA-bound transcription factors, and probably represent a general mechanism of transcriptional regulation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex.

In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which induces endocytosis of E-cadherin. With a modified yeast 2-hybrid system, we isolated Hakai, an E-cadherin binding protein, which we have identified as an E3 ubiquitin-ligase. Hakai contains SH2, RING, zinc-finger and proline-rich domains, and interacts with E-cadherin in a tyrosine phosphorylation-dependent manner, inducing ubiquitination of the E-cadherin complex. Expression of Hakai in epithelial cells disrupts cell--cell contacts and enhances endocytosis of E-cadherin and cell motility. Through dynamic recycling of E-cadherin, Hakai can thus modulate cell adhesion, and could participate in the regulation of epithelial--mesenchymal transitions in development or metastasis.

Amino Acid Sequence↗