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The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family (RTK) of transmembrane receptors, known to regulate many key cellular processes, including growth, proliferation, and differentiation. Its expression, activation, trafficking, and degradation have been extensively studied, as dysregulation of EGFR activation has been linked to a vast number of cancers. Activation of EGFR by different ligands results in distinct cellular responses, and the relative distribution of EGFR in different endosome pools in a process called endosomal sorting, leading to lysosomal degradation, or cell surface recycling, respectively, is considered a fundamental process in EGFR stimulation outcome. The EGFR interactome is therefore an essential element in the study of RTK functional selectivity. Here, we aimed to complement the existing EGFR interactome with spatio-temporal information on EGFR, its interactors, and phosphorylation state. We identified and quantified EGFR stable and transient interactions at different time points after stimulation using an EGFR-APEX2 fusion construct expressed in HEK293T cells and were able to use bystander proteins to map EGFR subcellular location at each time point. Utilizing the fast and concise biotinylation of proximity proteins by APEX2, we were able to detect slight differences in early signaling kinetics between TGF-α and EGF, thereby increasing our knowledge on RTK signaling and differential trafficking.

Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer. Hepatitis B virus encodes the regulatory HBx protein whose primary role is to promote transcription of the viral genome, which persists as an extrachromosomal DNA circle in infected cells. HBx accomplishes this task by an unusual mechanism, enhancing transcription only from extrachromosomal DNA templates. Here we show that HBx achieves this by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the 'structural maintenance of chromosomes' (Smc) complex Smc5/6 for degradation. Blocking this event inhibits the stimulatory effect of HBx both on extrachromosomal reporter genes and on hepatitis B virus transcription. Conversely, silencing the Smc5/6 complex enhances extrachromosomal reporter gene transcription in the absence of HBx, restores replication of an HBx-deficient hepatitis B virus, and rescues wild-type hepatitis B virus in a DDB1-knockdown background. The Smc5/6 complex associates with extrachromosomal reporters and the hepatitis B virus genome, suggesting a direct mechanism of transcriptional inhibition. These results uncover a novel role for the Smc5/6 complex as a restriction factor selectively blocking extrachromosomal DNA transcription. By destroying this complex, HBx relieves the inhibition to allow productive hepatitis B virus gene expression.

Breast cancer is the most prevalent type of malignancy in women with ∼1.7 million new cases diagnosed annually, of which the majority express ERα (ESR1), a ligand-dependent transcription factor. Genome-wide chromatin binding maps suggest that ERα may control the expression of thousands of genes, posing a great challenge in identifying functional targets. Recently, we developed a CRISPR-Cas9 functional genetic screening approach to identify enhancers required for ERα-positive breast cancer cell proliferation. We validated several candidates, including CUTE, a putative ERα-responsive enhancer located in the first intron of CUEDC1 (CUE-domain containing protein). Here, we show that CUTE controls CUEDC1 expression, and that this interaction is essential for ERα-mediated cell proliferation. Moreover, ectopic expression of CUEDC1, but not a CUE-domain mutant, rescues the defects in CUTE activity. Finally, CUEDC1 expression correlates positively with ERα in breast cancer. Thus, CUEDC1 is a functional target gene of ERα and is required for breast cancer cell proliferation.

Oncogene-induced senescence (OIS), provoked in response to oncogenic activation, is considered an important tumor suppressor mechanism. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt without a protein-coding capacity. Functional studies showed that deregulated lncRNA expression promote tumorigenesis and metastasis and that lncRNAs may exhibit tumor-suppressive and oncogenic function. Here, we first identified lncRNAs that were differentially expressed between senescent and non-senescent human fibroblast cells. Using RNA interference, we performed a loss-function screen targeting the differentially expressed lncRNAs, and identified lncRNA-OIS1 (lncRNA#32, AC008063.3 or ENSG00000233397) as a lncRNA required for OIS. Knockdown of lncRNA-OIS1 triggered bypass of senescence, higher proliferation rate, lower abundance of the cell-cycle inhibitor CDKN1A and high expression of cell-cycle-associated genes. Subcellular inspection of lncRNA-OIS1 indicated nuclear and cytosolic localization in both normal culture conditions as well as following oncogene induction. Interestingly, silencing lncRNA-OIS1 diminished the senescent-associated induction of a nearby gene (Dipeptidyl Peptidase 4, DPP4) with established role in tumor suppression. Intriguingly, similar to lncRNA-OIS1, silencing DPP4 caused senescence bypass, and ectopic expression of DPP4 in lncRNA-OIS1 knockdown cells restored the senescent phenotype. Thus, our data indicate that lncRNA-OIS1 links oncogenic induction and senescence with the activation of the tumor suppressor DPP4.

Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cancer development. HBV encodes the hepatitis B virus X (HBx) protein that promotes transcription of the viral episomal DNA genome by the host cell RNA polymerase II. Here we provide evidence that HBx accomplishes this task by a conserved and unusual mechanism. Thus, HBx strongly stimulates expression of transiently transfected reporter constructs, regardless of the enhancer and promoter sequences. This activity invariably requires HBx binding to the cellular UV-damaged DDB1 E3 ubiquitin ligase, suggesting a common mechanism. Unexpectedly, none of the reporters tested is stimulated by HBx when integrated into the chromosome, despite remaining responsive to their cognate activators. Likewise, HBx promotes gene expression from the natural HBV episomal template but not from a chromosomally integrated HBV construct. The same was observed with the HBx protein of woodchuck HBV. HBx does not affect nuclear plasmid copy number and functions independently of CpG dinucleotide methylation. Conclusion: We propose that HBx supports HBV gene expression by a conserved mechanism that acts specifically on episomal DNA templates independently of the nature of the cis-regulatory sequences. Because of its uncommon property and key role in viral transcription, HBx represents an attractive target for new antiviral therapies. (HEPATOLOGY 2012;56:2116–2124)

The cullin 4-DNA-damage-binding protein 1 (CUL4-DDB1) ubiquitin ligase machinery regulates diverse cellular functions and can be subverted by pathogenic viruses. Here we report the crystal structure of DDB1 in complex with a central fragment of hepatitis B virus X protein (HBx), whose DDB1-binding activity is important for viral infection. The structure reveals that HBx binds DDB1 through an alpha-helical motif, which is also found in the unrelated paramyxovirus SV5-V protein despite their sequence divergence. Our structure-based functional analysis suggests that, like SV5-V, HBx captures DDB1 to redirect the ubiquitin ligase activity of the CUL4-DDB1 E3 ligase. We also identify the alpha-helical motif shared by these viral proteins in the cellular substrate-recruiting subunits of the E3 complex, the DDB1-CUL4-associated factors (DCAFs) that are functionally mimicked by the viral hijackers. Together, our studies reveal a common yet promiscuous structural element that is important for the assembly of cellular and virally hijacked CUL4-DDB1 E3 complexes.

Many enteroviruses, members of the family Picornaviridae , cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3′ end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5′-end–3′-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.

Tumour growth and metabolic adaptation may restrict the availability of certain amino acids for protein synthesis. It has recently been shown that certain types of cancer cells depend on glycine, glutamine, leucine and serine metabolism to proliferate and survive. In addition, successful therapies using L-asparaginase-induced asparagine deprivation have been developed for acute lymphoblastic leukaemia. However, a tailored detection system for measuring restrictive amino acids in each tumour is currently not available. Here we harness ribosome profiling for sensing restrictive amino acids, and develop diricore, a procedure for differential ribosome measurements of codon reading. We first demonstrate the functionality and constraints of diricore using metabolic inhibitors and nutrient deprivation assays. Notably, treatment with L-asparaginase elicited both specific diricore signals at asparagine codons and high levels of asparagine synthetase (ASNS). We then applied diricore to kidney cancer and discover signals indicating restrictive proline. As for asparagine, this observation was linked to high levels of PYCR1, a key enzyme in proline production, suggesting a compensatory mechanism allowing tumour expansion. Indeed, PYCR1 is induced by shortage of proline precursors, and its suppression attenuated kidney cancer cell proliferation when proline was limiting. High PYCR1 is frequently observed in invasive breast carcinoma. In an in vivo model system of this tumour, we also uncover signals indicating restrictive proline. We further show that CRISPR-mediated knockout of PYCR1 impedes tumorigenic growth in this system. Thus, diricore has the potential to reveal unknown amino acid deficiencies, vulnerabilities that can be used to target key metabolic pathways for cancer treatment.