HeLa cDNA Library Research Articles

YY1AP, A Novel Co-activator of YY1

With the aim of identifying potential cellular proteins that mediate the transcriptional regulation of YY1, a HeLa cDNA library was screened using the yeast two-hybrid system. A previously unknown protein interacting with YY1 was identified and named YY1AP. By using the 5'-rapid amplification of cDNA ends technique, the full-length cDNA of YY1AP was cloned and sequenced. The cDNA was 2253 bp in length and encoded an open reading frame of 750 amino acids. The chromosomal gene was made up of 10 exons separated by nine introns and is localized on chromosome 1 (1q21.3). Northern blot analysis revealed that YY1AP is ubiquitously expressed in various human tissues and cancer cell lines. Co-immunoprecipitation and immunostaining of cells further indicated that YY1AP co-localizes with YY1 in the nucleus. Furthermore, YY1AP was shown to be capable of enhancing the transcriptional activation of an YY1 responsive promoter. Subsequent analysis by glutathione S-transferase pull-down assay showed that YY1AP contained two YY1 binding regions. The transactivation region of YY1AP would seem to be localized within the section of amino acids 260-345. It is proposed that YY1AP is a novel co-activator of YY1.

GTT1/StarD7, a Novel Phosphatidylcholine Transfer Protein-like Highly Expressed in Gestational Trophoblastic Tumour:: Cloning and Characterization

We report the cDNA cloning and characterization of GTT1/StarD7, a novel gestational trophoblastic tumour gene, initially identified by its up-regulated expression in the choriocarcinoma JEG-3 cell line with respect to their nonmalignant counterpart, complete hydatidiform mole and normal trophoblastic tissue. Using the differential display fragment as a probe we screened placenta and HeLa cDNA libraries and isolated a clone carrying a 3315 bp insert (accession number AF270647). This cDNA encodes a protein of 295 amino acid residues with a molecular weight of approximately 34.7 kDa and a pI of 5.79. Computer-mediated homology search revealed that the deduced amino acid sequence had similarity to phosphatidylcholine transfer protein (PCTP) with a conserved StAR-related lipid transfer (START) domain extending between the amino acids 66 to 250. The GTT1 gene contains at least 9 exons spread nearly 30 kb on chromosome 2p12-2p11.2. Northern blot assays of total RNA derived from normal early placenta (NEP), complete hydatidiform mole (CHM) and JEG-3 cell line revealed a 3.5 kb mRNA expressed exclusively in the JEG-3 cell line. However, semiquantitative RT–PCR analysis performed with the same RNA samples demonstrated GTT1 expression throughout all of them with the highest level in JEG-3 cell line. Examination of GTT1 mRNA expression by semiquantitative RT–PCR assays in a series of tumour cell lines indicated wide-spread GTT1 expression with predominance in both choriocarcinoma JEG-3 and JAR cells, colorectal adenocarcinoma HT29 and hepatocellular carcinoma HepG2 cells. In conclusion, the highly GTT1 expression profile in JEG-3 and JAR cell lines and its lipid binding domain suggest that GTT1 may play an important role in the phospholipid-mediated signalling of trophoblastic tumour cellular events.

Clinical and serological associations of autoantibodies to GW bodies and a novel cytoplasmic autoantigen GW182.

A novel autoantigen named GW182 was recently identified when the serum from a patient with a sensory ataxic polyneuropathy was used to immunoscreen a HeLa cDNA library. Unique features of the GW182 protein include 39 repeats of glycine (G) and tryptophan (W) residues, binding to a subset of messenger RNA and localization to unique structures within the cytoplasm that were designated GW bodies (GWBs). The goal of the present study was to identify the clinical features of patients with anti-GW182 antibodies and to characterize the B cell anti-GW182 response by defining the epitopes bound by human autoantibodies. The most common clinical diagnosis of patients with anti-GW182 antibodies was Sjögren's syndrome followed by mixed motor/sensory neuropathy, and systemic lupus erythematosus. Of interest, 5 (28%), 9 (50%), and 3 (17%) of the 18 sera that react with GWBs had autoantibodies to the GW182 and the 52 kDa and 60 kDa SS-A/Ro autoantigens, respectively. Epitopes bound by the human autoantibodies were mapped to the GW-rich middle part of the protein, the non-GW rich region, and the C-terminus of GW182 protein. None of the GW182 epitopes had significant sequence similarities to other known proteins. GW182 represents a new category of ribonucleoprotein autoantigens.

Molecular cloning of a novel human gene encoding histone acetyltransferase-like protein involved in transcriptional activation of hTERT

To isolate proteins involved in hTERT transcriptional regulation, the HeLa cDNA library was screened using the hTERT promoter-based yeast one-hybrid assay. A positive clone was rescued and proved to contain an open reading frame and the upstream coding sequences were obtained by 5 ′-RACE. The assembled full cDNA consisted of a 2.5 kb reading frame encoding 834 amino acids, in which a conserved N-acetyltransferase domain (GNAT family) was searched out in bioinformatics, and thus named as hALP (human N-acetyltransferase-like protein, GenBank Accession No. AF 489535). The expression of native hALP was identified in HeLa cells and proved to distribute in the cellular nucleus. The binding potential of hALP to hTERT promoter was confirmed by EMSA and the interacting sequence involved to −201- to −56-nt upstream region of the promoter. On transfection assay, hALP could obviously transactivate hTERT promoter and stimulate endogenous telomerase activity of cells. The analysis on histone acetyltransferase showed that hALP could specifically acetylate free histones in vitro. The investigation suggested that hALP influences the activity of histone acetylation and could up-regulate telomerase activity through transactivation of hTERT promoter.

Identification and characterization of NIF3L1 BP1, a novel cytoplasmic interaction partner of the NIF3L1 protein

The NIF3L1 protein is strongly conserved during evolution from bacteria to mammals and recently its function in neuronal differentiation has been demonstrated. In the present study we identified novel binding partners of human NIF3L1 by screening a HeLa cDNA-library using the yeast two-hybrid system. We could show that the NIF3L1 protein is interacting with itself and with the NIF3L1 binding protein 1 (NIF3L1 BP1), a novel protein of 23.67 kDa bearing a putative leucine zipper domain. Furthermore, both interactions were confirmed using the mammalian two-hybrid system. Deletion analyses clearly demonstrated that a C-terminal region of 100 amino acids of the NIF3L1 BP1 is sufficient for the interaction with NIF3L1. The NIF3L1 BP1 is ubiquitously expressed and cotransfection experiments revealed that NIF3L1 and NIF3L1 BP1 interact in the cytoplasm of human LNCaP cells. This study provides novel insights into the cellular function of the NIF3L1 protein.

Tumor necrosis factor receptor-associated factor (TRAF) 4 is a new binding partner for the p70S6 serine/threonine kinase

p70S6K is an intracellular serine/threonine kinase that mediates cell cycle progression and gene transcription. Immunofluorescent staining shows in factor-dependent hematopoietic M-07e cells that p70S6K is localized both in the cytosol and, after cytokine stimulation, also in the nucleus. We hypothesized that the p70S6K might interact with a transcription factor in the nucleus or with other proteins in the cytosol besides the S6 protein. By screening a yeast two-hybrid HeLa cDNA library with full-length p70S6K cDNA as bait, we identified tumor necrosis factor receptor-associated factor (TRAF) 4 as a new binding partner for this kinase. TRAF4 is a member of the TRAF family of putative signal-transducing proteins. Members of this family are capable of negatively regulating apoptotic pathways by inducing the expression of genes that promote cell survival. Immunoprecipitation experiments showed that stimulation of receptors of the tumor necrosis factor (TNF) family induced the formation of TRAF4/p70S6K complexes. Transfection studies showed that TRAF4 functions in p70S6K activation: TNF induced phosphorylation of S6, the main intracellular substrate of the kinase, in cells stably expressing TRAF4, but not in TRAF4-negative cells. In addition to its role in p70S6K activation, we postulate an anti-apoptotic role for TRAF4, because the agonistic anti-Fas antibody CH-11 induced apoptosis in untransfected HEK-293 cells, but not in TRAF4-expressing HEK-293 cells. In conclusion: (i) TNF-receptor activation leads to activation of the p70S6K; (ii) TRAF4 is a mediator in this TNF-induced signaling pathway; and (iii) TRAF4 inhibits Fas-induced apoptosis.

Identification of RanBMP interacting with Shigella flexneri IpaC invasin by two-hybrid system of yeast.

Bacillary dysentery caused by Shigella flexneri is still a threat to human health. Of four invasion plasmid antigen proteins (IpaA,B,C and D), IpaC plays an important role in the pathogenicity of this pathogen. The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease. By applying two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKT-ipaC. The bait plasmid was transformed AH109, and proved to express IpaC and then HeLa cDNA library plasmids were introduced into the above transformed AH109. The transformation mixture was plated on medium lacking Trp, Leu, and His in the initial screen, then restreaked on medium lacking Trp, Leu, His and Ade. Colonies growing on the selection medium were further assayed for beta-galactosidase activity. BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid. Among the 2X10(6) transformants, 64 positive clones were obtained as determined by activation of His, Ade and LacZ reporter genes. Sequence analysis revealed that cDNA inserts of two colonies were highly homologous to a known human protein, RanBPM. These results provide evidence that IpaC may be involved in the invasion process of S. flexneri by interacting with RanBPM, and RanBPM is most likely to be the downstream target of IpaC in the cascade events of S. flexneri infection.

Rab Coupling Protein (RCP), a Novel Rab4 and Rab11 Effector Protein

Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.

Hantavirus nucleocapsid protein interacts with the Fas-mediated apoptosis enhancer Daxx.

Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas. To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening. Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies. Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively. In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx. These data suggest a direct link between host cell machinery and a hantavirus structural component.

KRIT1 association with the integrin-binding protein ICAP-1: a new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis

Mutations in KRIT1, a protein initially identified based on a yeast two-hybrid interaction with the RAS-family GTPase RAP1A, are responsible for the development of the inherited vascular disorder cerebral cavernous malformations (CCM1). As the function of the KRIT1 protein and its role in CCM pathogenesis remain unknown, we performed yeast two-hybrid screens to identify additional protein binding partners. A fragment containing the N-terminal 272 amino acid residues of KRIT1, a region lacking similarity to any known protein upon database searches, was used as bait. From parallel screens of human fetal brain and HeLa cDNA libraries, we obtained multiple independent isolates of human integrin cytoplasmic domain-associated protein-1 (ICAP-1) as interacting clones. The interaction of KRIT1 and ICAP-1 was confirmed by GST-KRIT1 trapping of endogenous ICAP-1 from 293T cells. The alpha isoform of ICAP-1 is a 200 amino acid serine/threonine-rich phosphoprotein which binds the cytoplasmic tail of beta1 integrins. We show that mutagenesis of the N-terminal KRIT1 NPXY amino acid sequence, a motif critical for ICAP-1 binding to beta1 integrin molecules, completely abrogates the KRIT1/ICAP-1 interaction. The interaction between ICAP-1 and KRIT1, and the presence of a FERM domain in the latter, suggest that KRIT1 might be involved in the bidirectional signaling between integrin molecules and the cytoskeleton. Furthermore, these data suggest that KRIT1 might affect cell adhesion processes via integrin signaling in CCM1 pathogenesis.

Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38)

We have identified previously a new murine protein serine/threonine kinase, MPK38, closely related to the sucrose-non-fermenting protein kinase family [Gil, Yang, Lee, Choi and Ha (1997) Gene 195, 295–301]. Using the C-terminal half of the putative human counterpart of MPK38, HPK38, as a bait in a yeast two-hybrid screen of a human HeLa cDNA library, it was discovered that the zinc-finger-motif-containing protein, termed zinc-finger-like protein 9 (ZPR9), bound both HPK38 and MPK38. In a co-expression assay, ZPR9 associated with MPK38 in vivo, and we showed that the ZPR9 is also phosphorylated by MPK38. In addition, ZPR9 physically interacts with itself in mammalian cells. The ZPR9 cDNA hybridized with a mRNA species of approx. 1.7kb in Northern-blot analysis. The ZPR9 transcript was detected in all tissues examined, including lung, kidney, spleen, liver and brain. Co-expression of ZPR9 with MPK38 caused the accumulation of ZPR9 in the nucleus. These findings suggest a potentially important role for ZPR9 in MPK38-mediated signal transduction, and that ZPR9 is a physiological substrate of MPK38 in vivo.

Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38).

We have identified previously a new murine protein serine/threonine kinase, MPK38, closely related to the sucrose-non-fermenting protein kinase family [Gil, Yang, Lee, Choi and Ha (1997) Gene 195, 295-301]. Using the C-terminal half of the putative human counterpart of MPK38, HPK38, as a bait in a yeast two-hybrid screen of a human HeLa cDNA library, it was discovered that the zinc-finger-motif-containing protein, termed zinc-finger-like protein 9 (ZPR9), bound both HPK38 and MPK38. In a co-expression assay, ZPR9 associated with MPK38 in vivo, and we showed that the ZPR9 is also phosphorylated by MPK38. In addition, ZPR9 physically interacts with itself in mammalian cells. The ZPR9 cDNA hybridized with a mRNA species of approx. 1.7 kb in Northern-blot analysis. The ZPR9 transcript was detected in all tissues examined, including lung, kidney, spleen,liver and brain. Co-expression of ZPR9 with MPK38 caused the accumulation of ZPR9 in the nucleus. These findings suggest a potentially important role for ZPR9 in MPK38-mediated signal transduction, and that ZPR9 is a physiological substrate of MPK38 in vivo.

A Novel Transrepression Pathway of c-Myc: RECRUITMENT OF A TRANSCRIPTIONAL COREPRESSOR COMPLEX TO c-Myc BY MM-1, A c-Myc-BINDING PROTEIN

The protooncogene product c-Myc plays a role in transcription regulation both for activation and repression. While transactivation pathways of c-Myc either from the N-proximal or the C-proximal region that is linked to the chromatin remodeling complex have been identified, a transrepression pathway had been identified only from the C-proximal region via Max and Mad that recruit the histone deacetylase (HDAC) complex. We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc (Mori, K., Maeda, Y., Kitaura, H., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (1998) J. Biol. Chem. 273, 29794-29800). To clarify the molecular mechanisms of MM-1 toward c-Myc, cDNAs encoding MM-1-binding proteins were screened by the two-hybrid method with MM-1 as a bait using a human HeLa cDNA library, and a cDNA encoding TIF1 beta/KAP1, a transcriptional corepressor, was obtained. MM-1 was found to bind to the central portion of TIF1 beta in vitro and in vivo, and these proteins were found to be colocalized in the nucleus. MM-1 and TIF1 beta complex in human HeLa cells was found to also contain c-Myc, mSin3, and HDAC1. Introduction of the C-terminal half of TIF1 beta as a dominant negative form abrogated the inhibitory activity of MM-1 toward c-Myc and greatly stimulated the transcription activity of c-Myc. Moreover, the inhibitory activity of MM-1 toward c-Myc was canceled by trichostatin A, an inhibitor of HDAC1. These results indicate that MM-1 is a connecting factor that forms a novel transcription repression pathway of c-Myc.

The US2 gene product of herpes simplex virus type 2 interacts with cytokeratin 18.

In order to clarify the biological role of US2 gene product of herpes simplex virus type 2 (HSV-2), a HeLa cDNA library was screened in the yeast two-hybrid system using US2 protein as bait, and several interacting proteins were identified, including cytokeratin 18. US2 protein was co-immunoprecipitated with cytokeratin 18 from HSV-2 infected cell lysates. Analysis of infected orA431 cells by immunofluorescence showed that US2 protein gave filamentous or dot-like cytoplasmic staining pattern, and that it co-localized with cytokeratin 18. When US2 protein was expressed alone, it co-localized with cytokeratin 18. To define the domain interacting with cytokeratin 18, deletion mutant proteins were constructed and cells transfected with mutants were analyzed by indirect immunofluorescence. These results suggest that the N-terminal half of the US2 protein, especially the region containing amino acids 42-77, is important for interaction with cytokeratin 18.

AMY-1, a c-Myc-binding Protein, Is Localized in the Mitochondria of Sperm by Association with S-AKAP84, an Anchor Protein of cAMP-dependent Protein Kinase

We have reported that a novel c-Myc-binding protein, AMY-1 (associate of Myc-1), stimulated the transcription activity of c-Myc. To access the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human HeLa cDNA library, and a clone encoding cAMP-dependent protein kinase anchor protein 149 (AKAP149), was obtained. AMY-1 was found to bind in vitro and in vivo to the regulatory subunit II binding region of AKAP149 and S-AKAP84, a splicing variant of AKAP149 expressed in the testis. AMY-1 was expressed postmeiotically in the testis, as S-AKAP84 was expressed. Furthermore, S-AKAP84 and regulatory subunit II, a regulatory subunit of cAMP-dependent protein kinase, made a ternary complex in cells, and AMY-1 was localized in the mitochondria of HeLa and sperm in association with AKAP149 and S-AKAP84, respectively. These results suggest that AMY-1 plays a role in spermatogenesis.

Isolation and Characterization of a Tumor-Associated NADH Oxidase (tNOX) from the HeLa Cell Surface

Cell-surface-located, drug-responsive and tumor-associated NADH oxidase (tNOX) proteins were purified and characterized from HeLa cells. The proteins isolated exhibited NADH oxidase activity inhibited by capsaicin and were resistant to heating and to protease digestion. The activity was purified 200- to 500-fold to provide apparently homogeneous gel bands for N-terminal sequencing using three different protocols. All three protocols involved heat (50°C) and proteinase K treatment. Recovery of the total NADH oxidase activity was 86% and inhibition by capsaicin was 60 to 80%. After 450-fold purification, a 52-kDa component was obtained as a single gel band that retained the capsaicin-inhibitied NADH oxidase activity. Amino acid composition and partial amino acid sequences were obtained. The partial amino acid sequences were used to generate peptide antisera. Both the peptide antisera and polyclonal antisera to the 52-kDa component immunoprecipitated capsaicin-inhibited NADH oxidase activity and reacted with 52-, 34-, and 17-kDa components on Western blots from different steps of the purification. The tNOX protein exhibited immunological cross-reactivity and amino acid sequence identity with tNOX cloned from a HeLa cDNA library using a monoclonal antibody to tNOX from sera of cancer patients. The results provide a direct sequence link between tNOX of the HeLa cell surface and the cloned tNOX representative of patient sera. The tNOX form from the surface of HeLa cells yielded N-terminal sequence consistent with a coidentity of the cell surface and serum forms of the two activities.

A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA.

The xeroderma pigmentosum group A protein (XPA) plays a central role in nucleotide excision repair (NER). To identify proteins that bind to XPA, we screened a HeLa cDNA library using the yeast two-hybrid system. Here we report a novel cytoplasmic GTP-binding protein, designated XPA binding protein 1 (XAB1). The deduced amino acid sequence of XAB1 consisted of 374 residues with a molecular weight of 41 kDa and an isoelectric point of 4.65. Sequence analysis revealed that XAB1 has four sequence motifs G1-G4 of the GTP-binding protein family in the N-terminal half. XAB1 also contains an acidic region in the C-terminal portion. Northern blot analysis showed that XAB1 mRNA is expressed ubiquitously, and immunofluorescence analysis revealed that XAB1 is localized mainly in the cytoplasm. Consistent with the GTP-binding motif, purified recombinant XAB1 protein has intrinsic GTPase activity. Using the yeast two-hybrid system, we elucidated that XAB1 binds to the N-terminal region of XPA. The deletion of five amino acids, residues 30-34 of XPA, required for nuclear localization of XPA abolished the interaction with XAB1. These results suggest that XAB1 is a novel cytoplasmic GTPase involved in nuclear localization of XPA.

Human RNA lariat debranching enzyme cDNA complements the phenotypes of Saccharomyces cerevisiae dbr1 and Schizosaccharomyces pombe dbr1 mutants.

The cDNA encoding the human RNA lariat debranching enzyme (hDBR1) was identified and cloned by searching the Expressed Sequence Tag (EST) database and screening a HeLa cDNA library, based on predicted amino acid sequence homologies with the Saccharomyces cerevisiae, Schizosaccharomyces pombe and Caenorhabditis elegans debranching enzymes. The hDBR1 cDNA expressed in Escherichia coli showed debranching activity in vitro and was also shown to be functional in an interspecies specific complementation experiment. hDBR1 cDNA in a S. cerevisiae expression vector complemented the intron accumulation phenotype of a S. cerevisiae dbr1 null mutant. Integration of the cDNA for hDBR1 into the ura4 locus of S. pombe also complemented both the intron accumulation and slow growth phenotypes of a S. pombe dbr1 null mutant strain. Comparison of the amino acid sequence of hDBR1 with the other DBR protein sequences showed several conserved regions, with 40, 44 and 43% identity to the S. cerevisiae, S. pombe and C. elegans debranching enzymes, respectively.

Bovine Papillomavirus E1 Protein Is Sumoylated by the Host Cell Ubc9 Protein

Papillomavirus E1 protein is the replication initiator that recognizes and binds to the viral origin and initiates DNA strand separation through its ATP-dependent helicase activity. The E1 protein also functions in viral DNA replication by recruiting several cellular proteins to the origin, including host DNA polymerase alpha and replication protein A. To identify other cellular proteins that interact with bovine papillomavirus E1, an HeLa cDNA library was screened using a yeast two-hybrid assay. The host cell sumoylating enzyme, Ubc9, was found to interact specifically with E1 both in vitro and in vivo. Mapping studies localized critical E1 sequences for interaction to amino acids 315-459 and strongly implicated leucine 420 as critical for E1.Ubc9 complex formation. In addition to binding E1, Ubc9 catalyzed the covalent linkage of the ubiquitin-like protein, SUMO-1, to E1. An E1 mutant unable to bind Ubc9 showed normal intracellular stability, but was impaired for intranuclear distribution. Failure to accumulate in appropriate nuclear subdomains may account for the previously demonstrated replication defect of a human papillomavirus 16 E1 protein that was also unable to bind Ubc9 and suggests that sumoylation is a functionally important modification with regulatory implications for papillomavirus replication.

A Possible Interaction of Thioredoxin with VDUP1 in HeLa Cells Detected in a Yeast Two-Hybrid System

Human thioredoxin (hTrx), a small ubiquitous protein with strong reducing potential, has multiple biological functions, including signal transduction and regulation of the activity of transcription factors. hTrx expression is enhanced in HPV-transformed cancer cells; however, the role of hTrx in the malignant cells is not fully understood. We employed a yeast two-hybrid system to search for proteins that bind to hTrx in HeLa cells, a type of HPV-transformed human cervical cancer cell. In a screen of 1.62 × 106 yeast cotransformed with a HeLa cDNA library and an hTrx vector, 13 clones were identified as candidates for hTrx-binding proteins. Among them, 3 clones were found to code in frame for the carboxyl-terminal portion of VDUP1 protein, lacking at most the first 155 residues from the start codon. A reconstructed clone carrying the full-length VDUP1 coding sequence also showed the ability to bind to an hTrx fusion protein. Loss of interaction between VDUP1 and hTrx was observed either when two cysteines (Cys 32 and 35) in hTrx were substituted by serines or when the deletion in VDUP1 was extended from amino acid position 155 to 225 or beyond. The 71-mer peptide fragment (position 155–225) of VDUP-1 alone did not bind to hTrx.