HPV-18 E6 enhances the interaction between EMILIN2 and SNX27 to promote WNT signaling.

Oncogenic HPV E6 proteins have a PDZ-binding motif (PBM) which plays important roles in both the viral life cycle and tumor development. The PBM confers interaction with a large number of different PDZ domain-containing substrates, one of which is Sorting Nexin 27. This protein is part of the retromer complex and plays an important role in endocytic sorting pathways. It has been shown that at least two SNX27 interacting partners, GLUT1 and TANC2, are aberrantly trafficked due to the E6 PBM-dependent interaction with SNX27. To investigate further which other components of the endocytic trafficking pathway might be affected by the SNX27-HPV E6 interaction, we analyzed the SNX27 proteome interaction profile in a previously described HeLa cell line expressing GFP-SNX27, both in the presence and absence of the HPV-18 E6 oncoprotein. In this study, we identify a novel interacting partner of SNX27, secreted glycoprotein EMILIN2, whose release is blocked by HPV18 E6 in a PBM-dependent manner. Mechanistically, E6 can block EMILIN2 interaction with the WNT1 ligand, thereby enhancing WNT1 signaling and promoting cell proliferation. IMPORTANCE: This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.

HPV-18E6 Inhibits Interactions between TANC2 and SNX27 in a PBM-Dependent Manner and Promotes Increased Cell Proliferation.

Cancer-causing HPV E6 oncoproteins contain a PDZ-binding motif at the extreme carboxy terminus, which plays an important role in the viral life cycle and in the development of malignancy. Through this motif, HPV E6 targets a large number of cellular substrates, many of which are involved in processes related to the regulation of cell polarity. Recent studies also demonstrated E6's PDZ binding motif (PBM)-dependent association with SNX27, with a potential role in the perturbation of endocytic transport. Here, we have performed a proteomic analysis to identify SNX27-interacting partners whose binding to SNX27 is specifically perturbed in an E6-dependent manner. Extracts of HeLa cells that express GFP-tagged SNX27, transfected with control siRNA or siRNA targeting E6AP, were subject to GFP immunoprecipitation followed by mass spectroscopy, which identified TANC2 as an interacting partner of SNX27. Furthermore, we demonstrate that HPV E6 inhibits association between SNX27 and TANC2 in a PBM-dependent manner, resulting in an increase in TANC2 protein levels. In the absence of E6, SNX27 directs TANC2 toward lysosomal degradation. TANC2, in the presence of HPV-18E6, enhances cell proliferation in a PBM-dependent manner, indicating that HPV E6 targets the SNX27-mediated transport of TANC2 to promote cellular proliferation. IMPORTANCE While a great deal is known about the role of the E6 PDZ binding motif (PBM) in modulating the cellular proteins involved in regulating cell polarity, much less is known about the consequences of E6's interactions with SNX27 and the endocytic sorting machinery. We reasoned that a potential consequence of such interactions could be to affect the fate of multiple SNX27 endosomal partners, such as transmembrane proteins or soluble accessory proteins. Using a proteomic approach in HPV-18-positive cervical tumor-derived cells, we demonstrate that TANC2 is an interacting partner of SNX27, whose interaction is blocked by E6 in a PBM-dependent manner. This study therefore begins to shed new light on how E6 can regulate the endocytic transport of multiple SNX27-binding proteins, thereby expanding our understanding of the functions of the E6 PBM.

In Planta Colonization and Role of T6SS in Two Rice Kosakonia Endophytes.

Endophytes live inside plants and are often beneficial. Kosakonia is a novel bacterial genus that includes many diazotrophic plant-associated isolates. Plant-bacteria studies on two rice endophytic Kosakonia beneficial strains were performed, including comparative genomics, secretome profiling, in planta tests, and a field release trial. The strains are efficient rhizoplane and root endosphere colonizers and localized in the root cortex. Secretomics revealed 144 putative secreted proteins, including type VI secretory system (T6SS) proteins. A Kosakonia T6SS genomic knock-out mutant showed a significant decrease in rhizoplane and endosphere colonization ability. A field trial using rice seed inoculated with Kosakonia spp. showed no effect on plant growth promotion upon nitrogen stress and microbiome studies revealed that Kosakonia spp. were significantly more present in the inoculated rice. Comparative genomics indicated that several protein domains were enriched in plant-associated Kosakonia spp. This study highlights that Kosakonia is an important, recently classified genus involved in plant-bacteria interaction.

Phosphorylation of Human Papillomavirus Type 16 L2 Contributes to Efficient Virus Infectious Entry.

The human papillomavirus (HPV) capsid comprises two viral proteins, L1 and L2, with the L2 component being essential to ensure efficient endocytic transport of incoming viral genomes. Several studies have previously reported that L1 and L2 are posttranslationally modified, but it is uncertain whether these modifications affect HPV infectious entry. Using a proteomic screen, we identified a highly conserved phospho-acceptor site on the HPV-16 and bovine papillomavirus 1 (BPV-1) L2 proteins. The phospho-modification of L2 and its presence in HPV pseudovirions (PsVs) were confirmed using anti-phospho-L2-specific antibodies. Mutation of the phospho-acceptor sites of both HPV-16 and BPV-1 L2 resulted in the production of infectious virus particles, with no differences in efficiencies of packaging the reporter DNA. However, these mutated PsVs showed marked defects in infectious entry. Further analysis revealed a defect in uncoating, characterized by a delay in the exposure of a conformational epitope on L1 that indicates capsid uncoating. This uncoating defect was accompanied by a delay in the proteolysis of both L1 and L2 in mutated HPV-16 PsVs. Taken together, these studies indicate that phosphorylation of L2 during virus assembly plays an important role in optimal uncoating of virions during infection, suggesting that phosphorylation of the viral capsid proteins contributes to infectious entry.IMPORTANCE The papillomavirus L2 capsid protein plays an essential role in infectious entry, where it directs the successful trafficking of incoming viral genomes to the nucleus. However, nothing is known about how potential posttranslational modifications may affect different aspects of capsid assembly or infectious entry. In this study, we report the first phospho-specific modification of the BPV-1 and HPV-16 L2 capsid proteins. The phospho-acceptor site is very highly conserved across multiple papillomavirus types, indicating a highly conserved function within the L2 protein and the viral capsid. We show that this modification plays an essential role in infectious entry, where it modulates susceptibility of the incoming virus to capsid disassembly. These studies therefore define a completely new means of regulating the papillomavirus L2 proteins, a regulation that optimizes endocytic processing and subsequent completion of the infectious entry pathway.

LuxR Solos in the Plant Endophyte Kosakonia sp. Strain KO348.

Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization.IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.

New insight into antiphospholipid syndrome: antibodies to ß2glycoprotein I-domain 5 fail to induce thrombi in rats.

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of ß2glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analyzed the in vivo pro-coagulant effect of two groups of 5 sera IgG each reacting selectively with domain 1 or domain 5 in lipopolysaccharide (LPS)-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy, and vascular deposition of ß2glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-ß2glycoproteinI antibodies served as controls. All the anti-domain 1-positive IgG exhibited potent pro-coagulant activity while the anti-domain 5-positive and the negative control IgG failed to promote blood clot and vessel occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound ß2glycoproteinI while being able to interact with fluid-phase ß2glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit a thrombogenic effect in vivo, and the interaction of these antibodies with circulating ß2glycoproteinI suggests their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining "really at risk" patients for more appropriate treatments to avoid recurrences and disability.

A Broad Set of Chromatin Factors Influences Splicing.

Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing.

Analysis of Multiple HPV E6 PDZ Interactions Defines Type-Specific PDZ Fingerprints That Predict Oncogenic Potential.

The high-risk Human Papillomavirus (HPV) E6 oncoproteins are characterised by the presence of a class I PDZ-binding motif (PBM) on their extreme carboxy termini. The PBM is present on the E6 proteins derived from all cancer-causing HPV types, but can also be found on some related non-cancer-causing E6 proteins. We have therefore been interested in investigating the potential functional differences between these different E6 PBMs. Using an unbiased proteomic approach in keratinocytes, we have directly compared the interaction profiles of these different PBMs. This has allowed us to identify the potential PDZ target fingerprints of the E6 PBMs from 7 different cancer-causing HPV types, from 3 HPV types with weak cancer association, and from one benign HPV type that possesses an ancestral PBM. We demonstrate a striking increase in the number of potential PDZ targets bound by each E6 PBM as cancer-causing potential increases, and show that the HPV-16 and HPV-18 PBMs have the most flexibility in their PDZ target selection. Furthermore, the specific interaction with hScrib correlates directly with increased oncogenic potential. In contrast, hDlg is bound equally well by all the HPV E6 PBMs analysed, indicating that this is an evolutionarily conserved interaction, and was most likely one of the original E6 PBM target proteins that was important for the occupation of a potential new niche. Finally, we present evidence that the cell junction components ZO-2 and ß-2 syntrophin are novel PDZ domain-containing targets of a subset of high-risk HPV types.

The human papillomavirus E7 proteins associate with p190RhoGAP and alter its function.

UNLABELLED: Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis. IMPORTANCE: This study identifies p190RhoGAP as a novel cellular binding partner for the human papillomavirus (HPV) E7 protein. Our study shows that a large number of different HPV E7 proteins bind p190RhoGAP, and it identifies regions in both E7 and p190RhoGAP which are important for the interaction to occur. This study also highlights the likelihood that the E7-p190RhoGAP interaction may have important biological consequences related to actin organization in the infected cell. These changes could be an important contributor to the viral life cycle and during progression to cancer in HPV-infected cells. Importantly, this work also emphasizes the need for further study in a field which has largely been unexplored as it relates to the HPV life cycle and HPV-induced transformation.

PTMTreeSearch: a novel two-stage tree-search algorithm with pruning rules for the identification of post-translational modification of proteins in MS/MS spectra.

MOTIVATION: Tandem mass spectrometry has become a standard tool for identifying post-translational modifications (PTMs) of proteins. Algorithmic searches for PTMs from tandem mass spectrum data (MS/MS) tend to be hampered by noisy data as well as by a combinatorial explosion of search space. This leads to high uncertainty and long search-execution times. RESULTS: To address this issue, we present PTMTreeSearch, a new algorithm that uses a large database of known PTMs to identify PTMs from MS/MS data. For a given peptide sequence, PTMTreeSearch builds a computational tree wherein each path from the root to the leaves is labeled with the amino acids of a peptide sequence. Branches then represent PTMs. Various empirical tree pruning rules have been designed to decrease the search-execution time by eliminating biologically unlikely solutions. PTMTreeSearch first identifies a relatively small set of high confidence PTM types, and in a second stage, performs a more exhaustive search on this restricted set using relaxed search parameter settings. An analysis of experimental data shows that using the same criteria for false discovery, PTMTreeSearch annotates more peptides than the current state-of-the-art methods and PTM identification algorithms, and achieves this at roughly the same execution time. PTMTreeSearch is implemented as a plugable scoring function in the X!Tandem search engine. AVAILABILITY: The source code of PTMTreeSearch and a demo server application can be found at http://net.icgeb.org/ptmtreesearch

Characterizing TDP-43 interaction with its RNA targets.

One of the most important functional features of nuclear factor TDP-43 is its ability to bind UG-repeats with high efficiency. Several cross-linking and immunoprecipitation (CLIP) and RNA immunoprecipitation-sequencing (RIP-seq) analyses have indicated that TDP-43 in vivo can also specifically bind loosely conserved UG/GU-rich repeats interspersed by other nucleotides. These sequences are predominantly localized within long introns and in the 3'UTR of various genes. Most importantly, some of these sequences have been found to exist in the 3'UTR region of TDP-43 itself. In the TDP-43 3'UTR context, the presence of these UG-like sequences is essential for TDP-43 to autoregulate its own levels through a negative feedback loop. In this work, we have compared the binding of TDP-43 with these types of sequences as opposed to perfect UG-stretches. We show that the binding affinity to the UG-like sequences has a dissociation constant (Kd) of ∼110 nM compared with a Kd of 8 nM for straight UGs, and have mapped the region of contact between protein and RNA. In addition, our results indicate that the local concentration of UG dinucleotides in the CLIP sequences is one of the major factors influencing the interaction of these RNA sequences with TDP-43.

Human papillomavirus L2 facilitates viral escape from late endosomes via sorting nexin 17.

The human papillomavirus (HPV) L2 capsid protein plays an essential role during the early stages of viral infection, but the molecular mechanisms underlying its mode of action remain obscure. Using a proteomic approach, we have identified the adaptor protein, sorting nexin 17 (SNX17) as a strong interacting partner of HPV L2. This interaction occurs through a highly conserved SNX17 consensus binding motif, which is present in the majority of HPV L2 proteins analysed. Using mutants of L2 defective for SNX17 interaction, or siRNA ablation of SNX17 expression, we demonstrate that the interaction between L2 and SNX17 is essential for viral infection. Furthermore, loss of the L2-SNX17 interaction results in enhanced turnover of the L2 protein and decreased stability of the viral capsids, and concomitantly, there is a dramatic decrease in the efficiency with which viral genomes transit to the nucleus. Indeed, using a range of endosomal and lysosomal markers, we show that capsids defective in their capacity to bind SNX17 transit much more rapidly to the lysosomal compartment. These results demonstrate that the L2-SNX17 interaction is essential for viral infection and facilitates the escape of the L2-DNA complex from the late endosomal/lysosomal compartments.

Chemical rule-based filtering of MS/MS spectra.

MOTIVATION: Identification of proteins by mass spectrometry-based proteomics requires automated interpretation of peptide tandem mass spectrometry spectra. The effectiveness of peptide identification can be greatly improved by filtering out extraneous noise peaks before the subsequent database searching steps. RESULTS: Here we present a novel chemical rule-based filtering algorithm, termed CRF, which makes use of the predictable patterns (rules) of collision-induced peptide fragmentation. The algorithm selects peak pairs that obey the common fragmentation rules within plausible limits of mass tolerance as well as peak intensity and produces spectra that can be subsequently submitted to any search engine. CRF increases the positive predictive value and decreases the number of random matches and thus improves performance by 15-20% in terms of peptide annotation using search engines, such as X!Tandem. Importantly, the algorithm also achieves data compression rates of ∼75%. AVAILABILITY: The MATLAB source code and a web server are available at http://hydrax.icgeb.trieste.it/CRFilter/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The invasive capacity of HPV transformed cells requires the hDlg-dependent enhancement of SGEF/RhoG activity.

A major target of the HPV E6 oncoprotein is the human Discs Large (hDlg) tumour suppressor, although how this interaction contributes to HPV-induced malignancy is still unclear. Using a proteomic approach we show that a strong interacting partner of hDlg is the RhoG-specific guanine nucleotide exchange factor SGEF. The interaction between hDlg1 and SGEF involves both PDZ and SH3 domain recognition, and directly contributes to the regulation of SGEF's cellular localization and activity. Consistent with this, hDlg is a strong enhancer of RhoG activity, which occurs in an SGEF-dependent manner. We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity. In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6. Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells. These studies demonstrate that hDlg has a distinct oncogenic function in the context of HPV induced malignancy, one of the outcomes of which is increased RhoG activity and increased invasive capacity.

Pseudomonas fuscovaginae quorum sensing studies: 5% dominates cell-to-cell conversations.

A common feature of N-acyl-l-homoserine lactone (AHL) quorum-sensing (QS) systems is that the AHL signal is autoinducing. Once induced, a cell will further amplify the signal via a positive feedback loop. Pseudomonas fuscovaginae UPB0736 has two fully functional AHL QS systems, called PfsI/R and PfvI/R, which are inactive in a standard laboratory setting. In this work, we induce the QS systems with exogenous AHL signals and characterize the AHL signal amplification effect and QS activation dynamics at community and single-cell level. While the cognate signal is in both cases significantly further amplified to physiologically relevant levels, we observe only a limited response in terms of AHL synthase gene promoter activity. Additionally, the PfsI/R QS system exhibits a unique dramatic phenotypic heterogeneity, where only up to 5% of all cells amplify the signal further and are, thus, considered to be QS active. IMPORTANCE: Bacteria use N-acyl-l-homoserine lactone (AHL) quorum-sensing (QS) systems for population-wide phenotypic coordination. The QS configuration in Pseudomonas fuscovaginae is dramatically different from other model examples of AHL QS signaling and, thus, represents an important exception to the norm, which usually states that QS triggers population-wide phenotypic transitions in relation to cell density. We argue that the differences in QS dynamics of P. fuscovaginae highlight its different evolutionary purpose, which is ultimately dictated by the selective pressures of its natural habitat. We hope that this example will further expand our understanding of the complex and yet unknown QS-enabled sociomicrobiology. Furthermore, we argue that exemptions to the QS norm will be found in other plant-pathogenic bacterial strains that grow in similar environments and that molecularly similar QS systems do not necessarily share a similar evolutionary purpose; therefore, generalizations about bacterial cell-to-cell signaling systems function should be avoided.

HPV16 E7 modulates the cell surface expression of MET and CD109 via the AP2 complex.

Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein.

Functional analysis of the protein phosphatase activity of PTEN.

In vitro, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) displays intrinsic phosphatase activity towards both protein and lipid substrates. In vivo, the lipid phosphatase activity of PTEN, through which it dephosphorylates the 3 position in the inositol sugar of phosphatidylinositol derivatives, is important for its tumour suppressor function; however, the significance of its protein phosphatase activity remains unclear. Using two-photon laser-scanning microscopy and biolistic gene delivery of GFP (green fluorescent protein)-tagged constructs into organotypic hippocampal slice cultures, we have developed an assay of PTEN function in living tissue. Using this bioassay, we have demonstrated that overexpression of wild-type PTEN led to a decrease in spine density in neurons. Furthermore, it was the protein phosphatase activity, but not the lipid phosphatase activity, of PTEN that was essential for this effect. The ability of PTEN to decrease neuronal spine density depended upon the phosphorylation status of serine and threonine residues in its C-terminal segment and the integrity of the C-terminal PDZ-binding motif. The present study reveals a new aspect of the function of this important tumour suppressor and suggest that, in addition to dephosphorylating the 3 position in phosphatidylinositol phospholipids, the critical protein substrate of PTEN may be PTEN itself.

An analysis of CAF-1-interacting proteins reveals dynamic and direct interactions with the KU complex and 14-3-3 proteins.

CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly.

Regulation of the human papillomavirus type 18 E6/E6AP ubiquitin ligase complex by the HECT domain-containing protein EDD.

Human papillomavirus (HPV) E6 oncoproteins target many cellular proteins for ubiquitin-mediated proteasomal degradation. In the case of p53, this is mediated principally by the E6AP ubiquitin ligase. Several studies have reported that E6 can target certain of its substrates in an apparently E6AP-independent fashion and that several of these substrates vary in the degree to which they are degraded by E6 at different stages of malignancy. To more fully understand the regulation of the E6AP/E6 proteolytic targeting complex, we performed a mass spectroscopic analysis of HPV type 18 (HPV-18) E6 protein complexes and identified the HECT domain-containing ubiquitin ligase EDD as a new HPV-18 E6 binding partner. We show that EDD can interact independently with both E6 and E6AP. Furthermore, EDD appears to regulate E6AP expression levels independently of E6, and loss of EDD stimulates the proteolytic activity of the E6/E6AP complex. Conversely, higher levels of EDD expression protect a number of substrates from E6-induced degradation, partly as a consequence of lower levels of E6 and E6AP expression. Intriguingly, reduction in EDD expression levels in HPV-18-positive HeLa cells enhances cell resistance to apoptotic and growth arrest stimuli. These studies suggest that changes in the levels of EDD expression during different stages of the viral life cycle or during malignancy could have a profound effect upon the ability of E6 to target various substrates for proteolytic degradation and thereby directly influence the development of HPV-induced malignancy.

Alternative splicing regulation by interaction of phosphatase PP2Cgamma with nucleic acid-binding protein YB-1.

Kinases and phosphatases participate in precursor messenger RNA (pre-mRNA) splicing regulation, but their precise roles and the identities of their cofactors and substrates remain poorly understood. The human Ser/Thr phosphatase PP2Cgamma promotes spliceosome assembly. We show that PP2Cgamma's distinctive acidic domain is essential for its activity in splicing and interacts with YB-1, a spliceosome-associated factor. Moreover, PP2Cgamma is a phosphoprotein in vivo, and its acidic domain is phosphorylated under splicing conditions in vitro. PP2Cgamma phosphorylation enhances its interaction with YB-1 and is reversed by the phosphatase in cis. PP2Cgamma knockdown leaves constitutive splicing unaffected but inhibits cell proliferation and affects alternative splicing of CD44, a YB-1 target. This effect on splicing regulation is mediated by PP2Cgamma's acidic domain, which is essential to promote inclusion of CD44 exons v4 and v5 in vivo. We propose that PP2Cgamma modulates alternative splicing of specific pre-mRNAs coregulated by YB-1.