Role of glutaredoxin1 and glutathione in regulating the activity of the copper-transporting P-type ATPases, ATP7A and ATP7B.

The copper-transporting P-type ATPases (Cu-ATPases), ATP7A and ATP7B, are essential for the regulation of intracellular copper homeostasis. In this report we describe new roles for glutathione (GSH) and glutaredoxin1 (GRX1) in Cu homeostasis through their regulation of Cu-ATPase activity. GRX1 is a thiol oxidoreductase that catalyzes the reversible reduction of GSH-mixed disulfides to their respective sulfhydryls (deglutathionylation). Here, we demonstrated that glutathionylation of the Cu-ATPases and their interaction with GRX1 were affected by alterations in Cu levels. The data support our hypothesis that the Cu-ATPases serve as substrates for Cu-dependent GRX1-mediated deglutathionylation. This in turn liberates the Cu-ATPase cysteinyl thiol groups for Cu binding and transport. GSH depletion experiments led to reversible inhibition of the Cu-ATPases that correlated with effects on intracellular Cu levels and GRX1 activity. Finally, knockdown of GRX1 expression resulted in an increase in intracellular Cu accumulation. Together, these data directly implicate GSH and GRX1 with important new roles in redox regulation of the Cu-ATPases, through modulation of Cu binding by the Cu-ATPase cysteine motifs.

ZNF652, a novel zinc finger protein, interacts with the putative breast tumor suppressor CBFA2T3 to repress transcription.

The transcriptional repressor CBFA2T3 is a putative breast tumor suppressor. To define the role of CBFA2T3, we used a segment of this protein as bait in a yeast two-hybrid screen and identified a novel uncharacterized protein, ZNF652. In general, primary tumors and cancer cell lines showed lower expression of ZNF652 than normal tissues. Together with the location of this gene on the long arm of chromosome 17q, a region of frequent loss of heterozygosity in cancer, these results suggest a possible role of ZNF652 in tumorigenesis. In silico analysis of this protein revealed that it contains multiple classic zinc finger domains that are predicted to bind DNA. Coimmunoprecipitation assays showed that ZNF652 strongly interacts with CBFA2T3 and this interaction occurs through the COOH-terminal 109 amino acids of ZNF652. In contrast, there was a weak interaction of ZNF652 with CBFA2T1 and CBFA2T2, the other two members of this ETO family. Transcriptional reporter assays further confirmed the strength and selectivity of the ZNF652-CBFA2T3 interaction. The transcriptional repression of growth factor independent-1 (GFI-1), a previously characterized ETO effector zinc finger protein, was shown to be enhanced by CBFA2T1, but to a lesser extent by CBFA2T2 and CBFA2T3. We therefore suggest that each of the various gene effector zinc finger proteins may specifically interact with one or more of the ETO proteins to generate a defined range of transcriptional repressor complexes.

FBXO31 is the chromosome 16q24.3 senescence gene, a candidate breast tumor suppressor, and a component of an SCF complex.

A BAC located in the 16q24.3 breast cancer loss of heterozygosity region was previously shown to restore cellular senescence when transferred into breast tumor cell lines. We have shown that FBXO31, although located just distal to this BAC, can induce cellular senescence in the breast cancer cell line MCF-7 and is the likely candidate senescence gene. FBXO31 has properties consistent with a tumor suppressor, because ectopic expression of FBXO31 in two breast cancer cell lines inhibited colony growth on plastic and inhibited cell proliferation in the MCF-7 cell line. In addition, compared with the relative expression in normal breast, levels of FBXO31 were down-regulated in breast tumor cell lines and primary tumors. FBXO31 was cell cycle regulated in the breast cell lines MCF-10A and SKBR3 with maximal expression from late G(2) to early G(1) phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of cells at the G(1) phase of the cell cycle. FBXO31 contains an F-box domain and is associated with the proteins Skp1, Roc-1, and Cullin-1, suggesting that FBXO31 is a component of a SCF ubiquitination complex. We propose that FBXO31 functions as a tumor suppressor by generating SCF(FBXO31) complexes that target particular substrates, critical for the normal execution of the cell cycle, for ubiquitination and subsequent degradation.

CBFA2T3-ZNF652 corepressor complex regulates transcription of the E-box gene HEB.

Transcriptional repression plays a critical role in development and homeostasis. The ETO family represents a group of highly conserved and ubiquitously expressed transcriptional regulatory proteins that are components of a diverse range of multiprotein repressor complexes. ETO proteins function as transcriptional repressors by interacting with a number of transcription factors that bind to their cognate consensus DNA binding sequences within the promoters of target genes. We previously reported that the classical C(2)H(2) zinc finger DNA-binding protein, ZNF652, specifically and functionally interacts with the ETO protein CBFA2T3 and has a role in the suppression of breast oncogenesis. Here we report the identification and validation of the ZNF652 consensus DNA binding sequence. Our results show that the E-box gene HEB is a direct target of CBFA2T3-ZNF652-mediated transcriptional repression. The CBFA2T3-ZNF652 complex regulates HEB expression by binding to a single ZNF652 response element located within the promoter sequence of HEB. This study also shows that the NHR3 and NHR4 domains of CBFA2T3 interact with a conserved proline-rich region located within the C terminus of ZNF652. Our results, together with previous reports, indicate that HEB has a complex relationship with CBFA2T3; CBFA2T3 interacts with ZNF652 to repress HEB expression, and in addition CBFA2T3 interacts with the HEB protein to inhibit its activator function. These findings suggest that CBFA2T3-ZNF652-mediated HEB regulation may play an important role in hematopoiesis and myogenesis.