The cellular level of TRIM31, an RBCC protein overexpressed in gastric cancer, is regulated by multiple mechanisms including the ubiquitin-proteasome system.

TRIM (tripartite motif) family proteins, comprising RING finger, B-box and coiled-coil domains, are involved in various cellular processes including tumour development and antiviral response. One of the family proteins, TRIM31, was originally identified as a gene induced by growth-suppressive retinoid. Our previous study showed that TRIM31 is up-regulated in stomach cancer and that TRIM31 protein possesses the common features of the TRIM protein family, for example, ubiquitin ligase activity and homo-oligomerization tendency. Interestingly, TRIM31 negatively regulates growth of certain cell types despite its overexpression in gastric cancer tissues. We herein demonstrated that upon exogenous expression in 293 cells, TRIM31 is polyubiquitylated, which promotes its degradation in the proteasome pathway. The proteasome-mediated degradation of endogenous TRIM31 was further confirmed in AsPC-1 pancreatic cancer cells. Thus, this posttranslational modification governs the intracellular abundance of TRIM31, which is also dependent on inducible transcription as well as alternative splicing. The complicated control of the intracellular TRIM31 protein level may relate to its seemingly contradictory behaviours in the cancer pathology or the urgent response to viral infection.

Substrate-dependent metal preference of PPM1H, a cancer-associated protein phosphatase 2C: comparison with other family members.

Protein phosphatase 2C (PP2C) family is characterized by requirement of metal cation for phosphatase activity. We previously established that PPM1H is a cancer-associated member of the PP2C family. Here we further characterized the phosphatase activity of PPM1H, focusing on its dependence on metal cation. PPM1H possesses the potential to dephosphorylate p-nitrophenyl phosphate (pNPP), casein and phosphopeptides. Interestingly, PPM1H shows the metal preference that is varied depending on the substrate (substrate-dependent metal preference); PPM1H prefers Mn(2+) when pNPP or phosphopeptides is used as a substrate. Meanwhile, a preference for Mg(2+) is displayed by PPM1H with casein as a substrate. When both cations are added to the reaction, the degree of the effect is always closer to that with Mn(2+) alone, irrespective of the substrate. This preponderance of Mn(2+) is explained by its greater affinity for PPM1H than Mg(2+). From the literature the substrate-dependent metal preference appears to be shared by other PP2Cs. According to the crystal structure, a binuclear metal center of PP2C plays an important role for coordinating the substrate and nucleophilic waters in the active site. Therefore, the differences in the size, preferred geometry and coordination requirements between two metals, in relation to the substrate, may be responsible for this intriguing property.

Characterization of TRIM31, upregulated in gastric adenocarcinoma, as a novel RBCC protein.

To explore the molecules associated with gastric adenocarcinoma, we used the gene expression profile database of various human tissues and identified TRIM31 upregulated in both patients with chronic gastritis and stomach cancer. TRIM31 is a new member of RBCC proteins composed of RING finger, B-box and coiled-coil domains. We characterized TRIM31 biochemically and found it possess properties in common with other RBCC proteins, such as occurrence of alternative splicing transcripts, in vitro autoubiquitylating activity and a tendency to homo-oligomerize. The primary localization site of TRIM31 is the cytoplasm but some fraction is potentially associated with the mitochondria. TRIM31 overexpression suppresses colony formation of HCT116 cells while knockdown of its expression with short interfering RNAs (siRNAs) consistently tends to enhance growth of AsPC-1 cells slightly. Thus, TRIM31 is a characteristic RBCC protein with the ability to regulate cell proliferation negatively and may be a potential biomarker of gastric cancer as it is overexpressed from the early stage of gastric carcinogenesis.

Proteomic identification of a PSF/p54nrb heterodimer as RNF43 oncoprotein-interacting proteins.

RNF43 is an oncogenic RING finger protein overexpressed in colorectal cancer. To dissect its biological functions, we explored RNF43-interacting proteins by pull-down assay and MS. We identified a heterodimer, p54nrb and PSF, as RNF43's binding partners and confirmed their physical interaction in vivo by the co-immunoprecipitation experiment. Immunofluorescence analysis revealed that co-expression of PSF relocates RNF43 from the nuclear periphery to the nucleoplasm. Thus, proteomic identification of RNF43-associated proteins sheds light on its dynamic interaction network in nuclear events.

A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95.

RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with HAP95.

Protein phosphatase 1H, overexpressed in colon adenocarcinoma, is associated with CSE1L.

In search for a new anticancer drug target, we explored genes involved in colon adenocarcinoma development through dysregulation of a signal transduction pathway. By using the gene expression profile database, we found protein phosphatase 1H (PPM1H), belonging to the protein phosphatase 2C (PP2C) family, upregulated in colon adenocarcinomas compared with normal colon tissues. RT-PCR analysis verified the elevated level of PPM1H expression in colon cancer cell lines relative to a normal colon cell line. PPM1H encodes a protein with a molecular mass of approximately 50 kDa that resides in the cytoplasm. PPM1H fused with maltose-binding protein expressed in E. coli exhibited phosphatase activity characteristic of the PP2C family. Co-immunoprecipitation coupled with mass spectrometry analysis identified CSE1L, a proliferation and apoptosis-related protein, as a PPM1H-interacting protein. Native, but not inactive, PPM1H expressed in HeLa cells increased the mobility of CSE1L on SDS gels and a similar mobility shift was observed for purified CSE1L after treatment with PPM1H in vitro, supporting the notion that CSE1L is a substrate of PPM1H. Dominant negative PPM1H protected HeLa cells from cell death triggered by staurosporine or taxol. Additionally, knockdown of PPM1H expression with small interfering RNAs suppressed the growth of MCF-7 cells weakly but consistently. PPM1H controls cell cycle and proliferation of cancer cells potentially through dephosphorylation of CSE1L and might be a new target of anticancer drugs.

DDX39, upregulated in lung squamous cell cancer, displays RNA helicase activities and promotes cancer cell growth.

To explore differentially expressed genes involved in non-small cell lung cancer progression, we used the gene expression profile database of various human tissues and identified DDX39, a new member of the DEAD box RNA helicases, showing overexpression in human lung squamous cell carcinoma (LSCC) but not in lung adenocarcinoma (LAC). There existed three types of alternatively spliced DDX39 variants (DDX39-L, -S and -SS), of which only DDX39-L contains all the motifs required for RNA helicase activity. RT-PCR analysis verified the increased expression of DDX39-L in LSCC, but not LAC, cultured cells compared with normal bronchial epithelial cells. A high sequence similarity to UAP56 and punctate nuclear localization pattern of DDX39-L suggest that it plays a role in RNA splicing/export. Recombinant DDX39-L binds RNA, hydrolyzes NTPs in an RNA-dependent manner and unwinds double strand RNA bidirectionally, proving that DDX39 is an RNA helicase. Overexpression of DDX39-L stimulates colony formation of HeLa cells, probably through elevation of a translational level, indicating the biological significance of DDX39 in cancer pathogenesis. Thus, DDX39 is a novel RNA helicase capable of promoting cancer cell growth and, thereby, can be a potential target for development of a therapeutic strategy for LSCC.

Intracellular characterization of DDX39, a novel growth-associated RNA helicase.

DDX39 belongs to the DEAD box RNA helicase family and is overexpressed in human lung squamous cell carcinoma. In this study, in order to seek the biological relevance of DDX39, we conducted its intracellular characterization. When expressed in 293 cells, DDX39 undergoes heavy ubiquitylation and the stability of DDX39 is regulated via a ubiqutin-proteasome pathway. DDX39 tethers ALY, an essential mRNA export factor, in vivo, confirming the role of DDX39 in the RNA splicing/export process. Co-immunoprecipitation and mass spectrometry analyses detected CIP29, a recently discovered growth and cell cycle-related factor, as a main DDX39-interacting protein. CIP29 binds RNA on its own and enhances RNA unwinding activity of DDX39. Thus, CIP29 physically and functionally associates with DDX39, suggesting their cooperation in the RNA metabolism. Extension of the search for the protein-protein interactions encompassing DDX39 identified FUS/TLS, a nucleic acid binding protein participating in both transcription and splicing, as a CIP29-interacting protein. The connections comprising ALY, DDX39, CIP29 and FUS/TLS may be an integral part of transcription, splicing and RNA export. We simultaneously examined the properties of DDX39-S, a C-terminally truncated variant of DDX39 stemmed from alternative splicing, to understand its biological significance.

A novel mitochondrial C1-tetrahydrofolate synthetase is upregulated in human colon adenocarcinoma.

To seek the genes involved in the development of colorectal cancer, we analyzed the microarray gene expression profiles of human normal and cancerous colon tissues using the BioExpress database platform. Through the analysis we found one gene named DKFZp586G1517 that was upregulated in colon adenocarcinomas. The full-length cDNA of the DKFZp586G1517 cloned by polymerase chain reaction (PCR) encodes a protein with 978 amino acids, which is homologous to the human cytosolic C(1)-tetrahydrofolate synthetase and contains a mitochondrial target signal at N-terminus. The gene product expressed in 293 cells was localized in mitochondria and processed at the predicted signal cleavage site, supporting the idea that DKFZp586G1517 is a novel mitochondrial C(1)-tetrahydrofolate synthetase (mtC(1)-THFS). The overexpression of mtC(1)-THFS in 293 cells stimulated the colony formation. These results suggest that mtC(1)-THFS may participate in the progression of colorectal cancer by conferring growth advantage and could be a new molecular target for cancer therapy.

Baculoviral expression of correctly processed ADAMTS proteins fused with the human IgG-Fc region.

A disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) is a novel family of extracellular proteases supposedly involved in inflammation, angiogenesis, development and coagulation. To overexpress the active ADAMTS proteins, we designed a chimeric molecule composed of a catalytic domain of ADAMTS-1 or -4 and the human IgG Fc region in a baculoviral expression system. Both ADAMTS-Fc fusions were produced efficiently in the baculovirus-infected insect cells. The purified fusions underwent cleavage at the predicted furin recognition site. Both ADAMTS-Fc fusions bound to alpha(2)-macroglobulin, further indicating that they were correctly processed with the catalytic activity in this system; however, they failed to digest the peptides derived from the aggrecan sequences known to be clipped by the native enzyme, possibly due to the lack of required multiple interactions existing between the native protease and physiological substrate. In conclusion, the high productivity and facilitated purification of the fusion proteins would offer the source of the biochemical, biophysical or structural studies on the catalytic domain of the ADAMTS proteins.