Dong-Yan Jin

Regulatory Role for a Novel Human Thioredoxin Peroxidase in NF-κB Activation

Reduction-oxidation (redox) plays a critical role in NF-κB activation. Diverse stimuli appear to utilize reactive oxygen species (e.g. hydrogen peroxide) as common effectors for activating NF-κB. Antioxidants govern intracellular redox status, and many such molecules can reduce H2O2. However, functionally, it does appear that different antioxidants are variously selective for redox regulation of certain transcription factors such as NF-κB. For NF-κB, thioredoxin has been described to be a more potent antioxidant than either glutathione orN-acetylcysteine. Thioredoxin peroxidase is the immediate enzyme that links reduction of H2O2 to thioredoxin. Several putative human thioredoxin peroxidases have been identified using recursive sequence searches/alignments with yeast or prokaryotic enzymes. None has been characterized in detail for intracellular function(s). Here, we describe a new human thioredoxin peroxidase, antioxidant enzyme AOE372, identified by virtue of its protein-protein interaction with the product of a proliferation associationgene, pag, which is also a thiol-specific antioxidant. In human cells, AOE372 defines a redox pathway that specifically regulates NF-κB activity via a modulation of IκB-α phosphorylation in the cytoplasm. We show that AOE372 activity is regulated through either homo- or heterodimerization with other thiol peroxidases, implicating subunit assortment as a mechanism for regulating antioxidant specificities. AOE372 function suggests thioredoxin peroxidase as an immediate regulator of H2O2-mediated activation of NF-κB.

Role of Adapter Function in Oncoprotein-mediated Activation of NF-κB HUMAN T-CELL LEUKEMIA VIRUS TYPE I Tax INTERACTS DIRECTLY WITH IκB KINASE γ

Mechanisms by which the human T-cell leukemia virus type I Tax oncoprotein activates NF-κB remain incompletely understood. Although others have described an interaction between Tax and a holo-IκB kinase (IKK) complex, the exact details of protein-protein contact are not fully defined. Here we show that Tax binds to neither IKK-α nor IKK-β but instead complexes directly with IKK-γ, a newly characterized component of the IKK complex. This direct interaction with IKK-γ correlates with Tax-induced IκB-α phosphorylation and NF-κB activation. Thus, our findings establish IKK-γ as a key molecule for adapting an oncoprotein-specific signaling to IKK-α and IKK-β.

Hepatitis C virus core protein-induced loss of LZIP function correlates with cellular transformation

Hepatitis C virus (HCV) is the major etiological agent of blood‐borne non‐A non‐B hepatitis and a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. HCV core protein is a multifunctional protein with regulatory functions in cellular transcription and virus‐induced transformation and pathogenesis. Here we report on the identification of a bZIP nuclear transcription protein as an HCV core cofactor for transformation. This bZIP factor, designated LZIP, activates CRE‐dependent transcription and regulates cell proliferation. Loss of LZIP function in NIH 3T3 cells triggers morphological transformation and anchorage‐independent growth. We show that HCV core protein aberrantly sequesters LZIP in the cytoplasm, inactivates LZIP function and potentiates cellular transformation. Our findings suggest that LZIP might serve a novel cellular tumor suppressor function that is targeted by the HCV core.

Specific TATAA and bZIP requirements suggest that HTLV-I Tax has transcriptional activity subsequent to the assembly of an initiation complex

BackgroundHuman T-cell leukemia virus type I (HTLV-I) Tax protein is a transcriptional regulator of viral and cellular genes. In this study we have examined in detail the determinants for Tax-mediated transcriptional activation.ResultsWhereas previously the LTR enhancer elements were thought to be the sole Tax-targets, herein, we find that the core HTLV-I TATAA motif also provides specific responsiveness not seen with either the SV40 or the E1b TATAA boxes. When enhancer elements which can mediate Tax-responsiveness were compared, the authentic HTLV-I 21-bp repeats were found to be the most effective. Related bZIP factors such as CREB, ATF4, c-Jun and LZIP are often thought to recognize the 21-bp repeats equivalently. However, amongst bZIP factors, we found that CREB, by far, is preferred by Tax for activation. When LTR transcription was reconstituted by substituting either κB or serum response elements in place of the 21-bp repeats, Tax activated these surrogate motifs using surfaces which are different from that utilized for CREB interaction. Finally, we employed artificial recruitment of TATA-binding protein to the HTLV-I promoter in bypass experiments to show for the first time that Tax has transcriptional activity subsequent to the assembly of an initiation complex at the promoter.ConclusionsOptimal activation of the HTLV-I LTR by Tax specifically requires the core HTLV-I TATAA promoter, CREB and the 21-bp repeats. In addition, we also provide the first evidence for transcriptional activity of Tax after the recruitment of TATA-binding protein to the promoter.

17 – Peroxiredoxins in Cell Signaling and HIV Infection

This chapter summarizes the existing evidence and provides perspectives on the structure of peroxiredoxins as well as their functions in signal transduction and in HIV-1 infection. All peroxiredoxins conserve at least one cysteine motif at the N terminus. In addition to this N-terminal cysteine motif, many peroxiredoxins possess a second similar motif at the C terminus. These peroxiredoxin motifs are implicated as being important for the catalysis of peroxide reduction. This peroxidase activity, which accounts for the antioxidant properties of peroxiredoxins, is modulated by the microenvironment affecting the redox center residues. The redox status of the reactive center cysteine dictates the specificity and the potency of peroxiredoxin activity. The determination of a crystal structure of human provides a framework for understanding the mechanism and the regulation of peroxiredoxin functions. The ability of peroxiredoxins to reduce peroxides is attributed to their cysteine residues. The chapter presents models that are used to study the mechanisms by which peroxiredoxins reduce peroxides.

Cotranscriptional recruitment of yeast TRAMP complex to intronic sequences promotes optimal pre-mRNA splicing

Most unwanted RNA transcripts in the nucleus of eukaryotic cells, such as splicing-defective pre-mRNAs and spliced-out introns, are rapidly degraded by the nuclear exosome. In budding yeast, a number of these unwanted RNA transcripts, including spliced-out introns, are first recognized by the nuclear exosome cofactor Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex before subsequent nuclear-exosome-mediated degradation. However, it remains unclear when spliced-out introns are recognized by TRAMP, and whether TRAMP may have any potential roles in pre-mRNA splicing. Here, we demonstrated that TRAMP is cotranscriptionally recruited to nascent RNA transcripts, with particular enrichment at intronic sequences. Deletion of TRAMP components led to further accumulation of unspliced pre-mRNAs even in a yeast strain defective in nuclear exosome activity, suggesting a novel stimulatory role of TRAMP in splicing. We also uncovered new genetic and physical interactions between TRAMP and several splicing factors, and further showed that TRAMP is required for optimal recruitment of the splicing factor Msl5p. Our study provided the first evidence that TRAMP facilitates pre-mRNA splicing, and we interpreted this as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP before or during splicing to prepare for the subsequent targeting of spliced-out introns to rapid degradation by the nuclear exosome.

Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p

The heterogeneous nuclear ribonucleoprotein Npl3p of budding yeast is a substrate of arginine methyltransferase Hmt1p, but the role of Hmt1p in regulating Npl3p’s functions in transcription antitermination and elongation were unknown. We found that mutants lacking Hmt1p methyltransferase activity exhibit reduced recruitment of Npl3p, but elevated recruitment of a component of mRNA cleavage/termination factor CFI, to the activated GAL10-GAL7 locus. Consistent with this, hmt1 mutants displayed increased termination at the defective gal10-Δ56 terminator. Remarkably, hmt1Δ cells also exhibit diminished recruitment of elongation factor Tho2p and a reduced rate of transcription elongation in vivo. Importantly, the defects in Npl3p and Tho2p recruitment, antitermination and elongation in hmt1Δ cells all were mitigated by substitutions in Npl3p RGG repeats that functionally mimic arginine methylation by Hmt1p. Thus, Hmt1p promotes elongation and suppresses termination at cryptic terminators by methylating RGG repeats in Npl3p. As Hmt1p stimulates dissociation of Tho2p from an Npl3p-mRNP complex, it could act to recycle these elongation and antitermination factors back to sites of ongoing transcription.