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1.  A Broad Set of Chromatin Factors Influences Splicing 
PLoS Genetics  2016;12(9):e1006318.
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.
Author Summary
Splicing is an RNA editing step allowing to produce multiple transcripts from a single gene. The gene itself is organized in chromatin, associating DNA and multiple proteins. Some proteins regulating the compaction of the chromatin also affect RNA splicing. Yet, it was unclear whether these chromatin proteins were exceptions or whether chromatin very generally affected the outcome of splicing. Here, we show that a subset of chromatin proteins is physically in interaction with the enzyme responsible for RNA splicing. In addition, several chromatin proteins not found directly associated with the splicing machinery were also able to influence RNA splicing, suggesting that chromatin compaction very globally plays a role in splicing. This finding was confirmed using the first in vitro assay combining transcription and splicing in the context of chromatin; this assay showed that assembling DNA with chromatin proteins influences the efficiency of splicing.
PMCID: PMC5035054  PMID: 27662573
2.  Analysis of Multiple HPV E6 PDZ Interactions Defines Type-Specific PDZ Fingerprints That Predict Oncogenic Potential 
PLoS Pathogens  2016;12(8):e1005766.
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 β-2 syntrophin are novel PDZ domain–containing targets of a subset of high-risk HPV types.
Author Summary
The cancer-causing Human Papillomavirus (HPV) E6 oncoproteins have a unique carboxy terminal PDZ binding motif (PBM), which interacts with a number of different cellular PDZ domain-containing substrates. The PBM has important functions in both the viral life cycle and in HPV-induced malignancy. In this study we have used a proteomic approach to compare the ability of multiple HPV E6 oncoproteins to interact with different cellular PDZ proteins. We show a striking increase in the number of PDZ proteins recognised as the oncogenic potential of the individual E6 increases. Furthermore, we define combinations of PDZ proteins that are predictors of oncogenic potential, whilst others represent evolutionarily conserved targets bound across the evolutionary spectrum of low and high-risk PBM-containing E6 proteins. Taken together, these studies shed light on the functional conservation in the E6 PBM across multiple HPV types and support the hypothesis that ancestral PBM evolution originally conferred association with a restricted number of PDZ targets, which has, over time, evolved, to provide increased levels of flexibility and hence increase the number of cellular PDZ partners that can be bound by the cancer-causing E6 oncoproteins.
PMCID: PMC4970744  PMID: 27483446
3.  TRIM28 and β-Actin Identified via Nanobody-Based Reverse Proteomics Approach as Possible Human Glioblastoma Biomarkers 
PLoS ONE  2014;9(11):e113688.
Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.
PMCID: PMC4242679  PMID: 25419715
4.  The Human Papillomavirus E7 Proteins Associate with p190RhoGAP and Alter Its Function 
Journal of Virology  2014;88(7):3653-3663.
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.
PMCID: PMC3993551  PMID: 24403595
5.  CAPNS1 Regulates USP1 Stability and Maintenance of Genome Integrity 
Molecular and Cellular Biology  2013;33(12):2485-2496.
Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (μ-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Here we show that calpain is required for the stability of the deubiquitinating enzyme USP1 in several cell lines. USP1 modulates DNA replication polymerase choice and repair by deubiquitinating PCNA. The ubiquitinated form of the USP1 substrate PCNA is stabilized in CAPNS1-depleted U2OS cells and mouse embryonic fibroblasts (MEFs), favoring polymerase-η loading on chromatin and increased mutagenesis. USP1 degradation directed by the cell cycle regulator APC/Ccdh1, which marks USP1 for destruction in the G1 phase, is upregulated in CAPNS1-depleted cells. USP1 stability can be rescued upon forced expression of calpain-activated Cdk5/p25, previously reported as a cdh1 repressor. These data suggest that calpain stabilizes USP1 by activating Cdk5, which in turn inhibits cdh1 and, consequently, USP1 degradation. Altogether these findings point to a connection between the calpain system and the ubiquitin pathway in the regulation of DNA damage response and place calpain at the interface between cell cycle modulation and DNA repair.
PMCID: PMC3700094  PMID: 23589330
6.  Human Papillomavirus L2 facilitates viral escape from late endosomes via Sorting Nexin 17 
Traffic (Copenhagen, Denmark)  2012;13(3):455-467.
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.
PMCID: PMC3276720  PMID: 22151726
HPV; L2; viral infection; sorting nexin; endosomes
7.  Functional Analysis of the Protein Phosphatase Activity of PTEN 
Biochemical Journal  2012;444(3):457-464.
In vitro, the tumor suppressor PTEN 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 tumor suppressor function; however, the significance of its protein phosphatase activity remains unclear. Utilizing two-photon laser scanning microscopy and biolistic gene delivery of GFP-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 Ser and Thr residues in its C-terminal segment and the integrity of the C-terminal PDZ-binding motif. Our studies reveal a new aspect of the function of this important tumor suppressor and suggest that in addition to dephosphorylating the 3 position in phosphatidylinositol phospholipids, the critical protein substrate of PTEN may be PTEN itself.
PMCID: PMC3365644  PMID: 22413754
PTEN; protein phosphatase; two-photon laser scanning microscopy; organotypic; spine density; PDZ domain
8.  Functional analysis of the protein phosphatase activity of PTEN 
Biochemical Journal  2012;444(Pt 3):457-464.
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.
PMCID: PMC3365644  PMID: 22413754
organotypic hippocampal slice culture; PDZ domain; phosphatase and tensin homologue deleted on chromosome 10 (PTEN); protein phosphatase; spine density; two-photon laser-scanning microscopy; DsRed, Discosoma red fluorescent protein; DTT, dithiothreitol; GFP, green fluorescent protein; HEK, human embryonic kidney; NMDA, N-methyl-D-aspartate; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; PTP, protein tyrosine phosphatase
9.  The Invasive Capacity of HPV Transformed Cells Requires the hDlg-Dependent Enhancement of SGEF/RhoG Activity 
PLoS Pathogens  2012;8(2):e1002543.
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.
Author Summary
The E6 oncoproteins from cancer-causing Human Papillomaviruses (HPVs) all share the capacity to target cellular PDZ domain containing proteins. The first such target of E6 to be identified was the cell polarity regulator Discs Large (Dlg). However owing to the limited information on the molecular basis for hDlg function, there is currently no information on what the role of the HPV E6-Dlg interaction might mean for the development of cervical cancer. In this study we have identified the molecular basis by which Dlg can regulate cell migration and invasion. This involves an interaction with SGEF, which in turn results in enhanced levels of RhoG activity and hence increased cell invasive capacity. Most importantly, we show that HPV-18 and HPV-16 E6 maintain this activity of Dlg to enhance a cell's invasive potential. These studies have major implications for how E6-PDZ interactions might contribute to HPV induced malignancy. Furthermore they also provide compelling evidence to explain how Dlg can contribute to the regulation of cell invasion and migration, and indicate that certain cellular pools of Dlg have distinct oncogenic potential.
PMCID: PMC3285591  PMID: 22383878
10.  Regulation of the Human Papillomavirus Type 18 E6/E6AP Ubiquitin Ligase Complex by the HECT Domain-Containing Protein EDD▿  
Journal of Virology  2011;85(7):3120-3127.
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.
PMCID: PMC3067830  PMID: 21228227
11.  Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function 
Retrovirology  2011;8:60.
Central to the fully competent replication cycle of the human immunodeficiency virus type 1 (HIV-1) is the nuclear export of unspliced and partially spliced RNAs mediated by the Rev posttranscriptional activator and the Rev response element (RRE).
Here, we introduce a novel method to explore the proteome associated with the nuclear HIV-1 RNAs. At the core of the method is the generation of cell lines harboring an integrated provirus carrying RNA binding sites for the MS2 bacteriophage protein. Flag-tagged MS2 is then used for affinity purification of the viral RNA. By this approach we found that the viral RNA is associated with the host nuclear matrix component MATR3 (Matrin 3) and that its modulation affected Rev activity. Knockdown of MATR3 suppressed Rev/RRE function in the export of unspliced HIV-1 RNAs. However, MATR3 was able to associate with Rev only through the presence of RRE-containing viral RNA.
In this work, we exploited a novel proteomic method to identify MATR3 as a cellular cofactor of Rev activity. MATR3 binds viral RNA and is required for the Rev/RRE mediated nuclear export of unspliced HIV-1 RNAs.
PMCID: PMC3160904  PMID: 21771346
12.  Voltage sensor mutations differentially target misfolded K+ channel subunits to proteasomal and non-proteasomal disposal pathways 
FEBS letters  2004;568(1-3):110-116.
In Shaker K+ channels, formation of an electrostatic interaction between two charged residues, D316 and K374 in transmembrane segments S3 and S4, respectively, is a key step in voltage sensor biogenesis. Mutations D316K and K374E disrupt formation of the voltage sensor and lead to endoplasmic reticulum retention. We have now investigated the fates of these misfolded proteins. Both are significantly less stable than the wild-type protein. D316K is degraded by cytoplasmic proteasomes, whereas K374E is degraded by a lactacystin-insensitive, non-proteasomal pathway. Our results suggest that the D316K and K374E proteins are misfolded in recognizably different ways, an observation with implications for voltage sensor biogenesis.
PMCID: PMC3101709  PMID: 15196930
Shaker channel; Quality control; Degradation; Biogenesis
13.  Arginine Methylation Controls the Subcellular Localization and Functions of the Oncoprotein Splicing Factor SF2/ASF▿ †  
Molecular and Cellular Biology  2010;30(11):2762-2774.
Alternative splicing and posttranslational modifications (PTMs) are major sources of protein diversity in eukaryotic proteomes. The SR protein SF2/ASF is an oncoprotein that functions in pre-mRNA splicing, with additional roles in other posttranscriptional and translational events. Functional studies of SR protein PTMs have focused exclusively on the reversible phosphorylation of Ser residues in the C-terminal RS domain. We confirmed that human SF2/ASF is methylated at residues R93, R97, and R109, which were identified in a global proteomic analysis of Arg methylation, and further investigated whether these methylated residues regulate the properties of SF2/ASF. We show that the three arginines additively control the subcellular localization of SF2/ASF and that both the positive charge and the methylation state are important. Mutations that block methylation and remove the positive charge result in the cytoplasmic accumulation of SF2/ASF. The consequent decrease in nuclear SF2/ASF levels prevents it from modulating the alternative splicing of target genes, results in higher translation stimulation, and abrogates the enhancement of nonsense-mediated mRNA decay. This study addresses the mechanisms by which Arg methylation and the associated positive charge regulate the activities of SF2/ASF and emphasizes the significance of localization control for an oncoprotein with multiple functions in different cellular compartments.
PMCID: PMC2876523  PMID: 20308322
14.  The pseudophosphatase MK-STYX interacts with G3BP and decreases stress granule formation 
Biochemical Journal  2010;427(Pt 3):349-357.
MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein] is a pseudophosphatase member of the dual-specificity phosphatase subfamily of the PTPs (protein tyrosine phosphatases). MK-STYX is catalytically inactive due to the absence of two amino acids from the signature motif that are essential for phosphatase activity. The nucleophilic cysteine residue and the adjacent histidine residue, which are conserved in all active dual-specificity phosphatases, are replaced by serine and phenylalanine residues respectively in MK-STYX. Mutations to introduce histidine and cysteine residues into the active site of MK-STYX generated an active phosphatase. Using MS, we identified G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1], a regulator of Ras signalling, as a binding partner of MK-STYX. We observed that G3BP1 bound to native MK-STYX; however, binding to the mutant catalytically active form of MK-STYX was dramatically reduced. G3BP1 is also an RNA-binding protein with endoribonuclease activity that is recruited to ‘stress granules’ after stress stimuli. Stress granules are large subcellular structures that serve as sites of mRNA sorting, in which untranslated mRNAs accumulate. We have shown that expression of MK-STYX inhibited stress granule formation induced either by aresenite or expression of G3BP itself; however, the catalytically active mutant MK-STYX was impaired in its ability to inhibit G3BP-induced stress granule assembly. These results reveal a novel facet of the function of a member of the PTP family, illustrating a role for MK-STYX in regulating the ability of G3BP1 to integrate changes in growth-factor stimulation and environmental stress with the regulation of protein synthesis.
PMCID: PMC2873733  PMID: 20180778
dual-specificity phosphatase; mitogen-activated protein kinase serine-; threonine- and tyrosine-specific phosphatase (MK-STYX); pseudophosphatase; Ras-GTPase-activating protein Src-homology-3-domain-binding protein-1 (G3BP1); stress granule; CMT, Charcot–Marie–Tooth; CRHSP-24, calcium–responsive heat-stable protein with a molecular mass of 24 kDa; Cy3, indocarbocyanine; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; D(U)SP, dual-specificity phosphatase; eIF, eukaryotic translation initiation factor; FBS, fetal bovine serum; FLI1, Friend leukaemia virus integration 1; GAP, GTPase-activating protein; G3BP, Ras-GAP SH3-domain-binding protein; GFP, green fluorescent protein; HEK-293 cell; human embryonic kidney cell; IA2, islet cell antigen 512; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MKP, MAPK phosphatase; (MK-)STYX, (MAPK) phospho-serine/threonine/tyrosine-binding protein; MTM, myotubularin; MTMR, MTM-related protein; NA, numerical aperture; NTF, nuclear transport factor; p, phospho-; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; RPTP, receptor PTP; SH3, Src homology 3
15.  Role of the HCF-1 Basic Region in Sustaining Cell Proliferation 
PLoS ONE  2010;5(2):e9020.
The human herpes simplex virus-associated host cell factor 1 (HCF-1) is a conserved human transcriptional co-regulator that links positive and negative histone modifying activities with sequence-specific DNA-binding transcription factors. It is synthesized as a 2035 amino acid precursor that is cleaved to generate an amino- (HCF-1N) terminal subunit, which promotes G1-to-S phase progression, and a carboxy- (HCF-1C) terminal subunit, which controls multiple aspects of cell division during M phase. The HCF-1N subunit contains a Kelch domain that tethers HCF-1 to sequence-specific DNA-binding transcription factors, and a poorly characterized so called “Basic” region (owing to a high ratio of basic vs. acidic amino acids) that is required for cell proliferation and has been shown to associate with the Sin3 histone deacetylase (HDAC) component. Here we studied the role of the Basic region in cell proliferation and G1-to-S phase transition assays.
Methodology/Principal Findings
Surprisingly, much like the transcriptional activation domains of sequence-specific DNA-binding transcription factors, there is no unique sequence within the Basic region required for promoting cell proliferation or G1-to-S phase transition. Indeed, the ability to promote these activities is size dependent such that the shorter the Basic region segment the less activity observed. We find, however, that the Basic region requirements for promoting cell proliferation in a temperature-sensitive tsBN67 cell assay are more stringent than for G1-to-S phase progression in an HCF-1 siRNA-depletion HeLa-cell assay. Thus, either half of the Basic region alone can support G1-to-S phase progression but not cell proliferation effectively in these assays. Nevertheless, the Basic region displays considerable structural plasticity because each half is able to promote cell proliferation when duplicated in tandem. Consistent with a potential role in promoting cell-cycle progression, the Sin3a HDAC component can associate independently with either half of the Basic region fused to the HCF-1 Kelch domain.
While conserved, the HCF-1 Basic region displays striking structural flexibility for controlling cell proliferation.
PMCID: PMC2814863  PMID: 20126307
16.  Molecular Mechanisms of Paraptosis Induction: Implications for a Non-Genetically Modified Tumor Vaccine 
PLoS ONE  2009;4(2):e4631.
Paraptosis is the programmed cell death pathway that leads to cellular necrosis. Previously, rodent and human monocytes/macrophages killed glioma cells bearing the membrane macrophage colony stimulating factor (mM-CSF) through paraptosis, but the molecular mechanism of this killing process was never identified. We have demonstrated that paraptosis of rat T9 glioma cells can be initiated through a large potassium channel (BK)-dependent process initiated by reactive oxygen species. Macrophage mediated cytotoxicity upon the mM-CSF expressing T9-C2 cells was not prevented by the addition of the caspase inhibitor, zVAD-fmk. By a combination of fluorescent confocal and electron microscopy, flow cytometry, electrophysiology, pharmacology, and genetic knock-down approaches, we demonstrated that these ion channels control cellular swelling and vacuolization of rat T9 glioma cells. Cell lysis is preceded by a depletion of intracellular ATP. Six-hour exposure to BK channel activation caused T9 cells to over express heat shock proteins (Hsp 60, 70, 90 and gp96). This same treatment forced HMGB1 translocation from the nuclear region to the periphery. These last molecules are “danger signals” that can stimulate immune responses. Similar inductions of mitochondrial swelling and increased Hsp70 and 90 expressions by BK channel activation were observed with the non-immunogenic F98 glioma cells. Rats injected with T9 cells which were killed by prolonged BK channel activation developed immunity against the T9 cells, while the injection of x-irradiated apoptotic T9 cells failed to produce the vaccinating effect. These results are the first to show that glioma cellular death induced by prolonged BK channel activation improves tumor immunogenicity; this treatment reproduces the vaccinating effects of mM-CSF transduced cells. Elucidation of strategies as described in this study may prove quite valuable in the development of clinical immunotherapy against cancer.
PMCID: PMC2645013  PMID: 19247476
17.  Control of Pre-mRNA Splicing by the General Splicing Factors PUF60 and U2AF65 
PLoS ONE  2007;2(6):e538.
Pre-mRNA splicing is a crucial step in gene expression, and accurate recognition of splice sites is an essential part of this process. Splice sites with weak matches to the consensus sequences are common, though it is not clear how such sites are efficiently utilized. Using an in vitro splicing-complementation approach, we identified PUF60 as a factor that promotes splicing of an intron with a weak 3′ splice-site. PUF60 has homology to U2AF65, a general splicing factor that facilitates 3′ splice-site recognition at the early stages of spliceosome assembly. We demonstrate that PUF60 can functionally substitute for U2AF65 in vitro, but splicing is strongly stimulated by the presence of both proteins. Reduction of either PUF60 or U2AF65 in cells alters the splicing pattern of endogenous transcripts, consistent with the idea that regulation of PUF60 and U2AF65 levels can dictate alternative splicing patterns. Our results indicate that recognition of 3′ splice sites involves different U2AF-like molecules, and that modulation of these general splicing factors can have profound effects on splicing.
PMCID: PMC1888729  PMID: 17579712
18.  Regulation of Adeno-Associated Virus DNA Replication by the Cellular TAF-I/Set Complex†  
Journal of Virology  2006;80(14):6855-6864.
The Rep proteins of the adeno-associated virus (AAV) are required for viral replication in the presence of adenovirus helper functions and as yet poorly characterized cellular factors. In an attempt to identify such factors, we purified Flag-Rep68-interacting proteins from human cell lysates. Several polypeptides were identified by mass spectrometry, among which was ANP32B, a member of the acidic nuclear protein 32 family which takes part in the formation of the template-activating factor I/Set oncoprotein (TAF-I/Set) complex. The N terminus of Rep was found to specifically bind the acidic domain of ANP32B; through this interaction, Rep was also able to recruit other members of the TAF-I/Set complex, including the ANP32A protein and the histone chaperone TAF-I/Set. Further experiments revealed that silencing of ANP32A and ANP32B inhibited AAV replication, while overexpression of all of the components of the TAF-I/Set complex increased de novo AAV DNA synthesis in permissive cells. Besides being the first indication that the TAF-I/Set complex participates in wild-type AAV replication, these findings have important implications for the generation of recombinant AAV vectors since overexpression of the TAF-I/Set components was found to markedly increase viral vector production.
PMCID: PMC1489034  PMID: 16809291
19.  A primate virus generates transformed human cells by fusion 
The Journal of Cell Biology  2005;171(3):493-503.
Amodel that explains both the origin and sporadic nature of cancer argues that cancer cells are a chance result of events that cause genomic and epigenetic variability. The prevailing view is that these events are mutations that affect chromosome segregation or stability. However, genomic and epigenetic variability is also triggered by cell fusion, which is often caused by viruses. Yet, cells fused by viruses are considered harmless because they die. We provide evidence that a primate virus uses both viral and exosomal proteins involved in cell fusion to produce transformed proliferating human cells. Although normal cells indeed fail to proliferate after fusion, expression of an oncogene or a mutated tumor suppressor p53 in just one of the fusion partners is sufficient to produce heterogeneous progeny. We also show that this virus can produce viable oncogenically transformed cells by fusing cells that are otherwise destined to die. Therefore, we argue that viruses can contribute to carcinogenesis by fusing cells.
PMCID: PMC2171256  PMID: 16275753
20.  Characterization of Human RNA Polymerase III Identifies Orthologues for Saccharomyces cerevisiae RNA Polymerase III Subunits 
Molecular and Cellular Biology  2002;22(22):8044-8055.
Unlike Saccharomyces cerevisiae RNA polymerase III, human RNA polymerase III has not been entirely characterized. Orthologues of the yeast RNA polymerase III subunits C128 and C37 remain unidentified, and for many of the other subunits, the available information is limited to database sequences with various degrees of similarity to the yeast subunits. We have purified an RNA polymerase III complex and identified its components. We found that two RNA polymerase III subunits, referred to as RPC8 and RPC9, displayed sequence similarity to the RNA polymerase II RPB7 and RPB4 subunits, respectively. RPC8 and RPC9 associated with each other, paralleling the association of the RNA polymerase II subunits, and were thus paralogues of RPB7 and RPB4. Furthermore, the complex contained a prominent 80-kDa polypeptide, which we called RPC5 and which corresponded to the human orthologue of the yeast C37 subunit despite limited sequence similarity. RPC5 associated with RPC53, the human orthologue of S. cerevisiae C53, paralleling the association of the S. cerevisiae C37 and C53 subunits, and was required for transcription from the type 2 VAI and type 3 human U6 promoters. Our results provide a characterization of human RNA polymerase III and show that the RPC5 subunit is essential for transcription.
PMCID: PMC134740  PMID: 12391170
21.  HIV-1 Nef Binds the DOCK2–ELMO1 Complex to Activate Rac and Inhibit Lymphocyte Chemotaxis 
PLoS Biology  2004;2(1):e6.
The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.
Upon HIV-1 infection of T cells, Nef activates Rac by binding the DOCK2-ELMO1 complex. In this way, Nef influences multiple aspects of T cell function, including inhibition of lymphocyte chemotaxis
PMCID: PMC314466  PMID: 14737186

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