A comparative analysis of Smad-responsive motifs identifies multiple regulatory inputs for TGF-ß transcriptional activation.

Smad proteins are transcriptional regulators activated by TGF-ß. They are known to bind to two distinct Smad-responsive motifs, namely the Smad-binding element (SBE) (5'-GTCTAGAC-3') and CAGA motifs (5'-AGCCAGACA-3' or 5'-TGTCTGGCT-3'). However, the mechanisms by which these motifs promote Smad activity are not fully elucidated. In this study, we performed DNA CASTing, binding assays, ChIP sequencing, and quantitative RT-PCR to dissect the details of Smad binding and function of the SBE and CAGA motifs. We observed a preference for Smad3 to bind CAGA motifs and Smad4 to bind SBE, and that either one SBE or a triple-CAGA motif forms a cis-acting functional half-unit for Smad-dependent transcription activation; combining two half-units allows efficient activation. Unexpectedly, the extent of Smad binding did not directly correlate with the abilities of Smad-binding sequences to induce gene expression. We found that Smad proteins are more tolerant of single bp mutations in the context of the CAGA motifs, with any mutation in the SBE disrupting function. CAGA and CAGA-like motifs but not SBE are widely distributed among stimulus-dependent Smad2/3-binding sites in normal murine mammary gland epithelial cells, and the number of CAGA and CAGA-like motifs correlates with fold-induction of target gene expression by TGF-ß. These data, demonstrating Smad responsiveness can be tuned by both sequence and number of repeats, provide a compelling explanation for why CAGA motifs are predominantly used for Smad-dependent transcription activation in vivo.

HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-ß type I receptor.

Viruses sometimes mimic host proteins and hijack the host cell machinery. Hepatitis C virus (HCV) causes liver fibrosis, a process largely mediated by the overexpression of transforming growth factor (TGF)-ß and collagen, although the precise underlying mechanism is unknown. Here, we report that HCV non-structural protein 3 (NS3) protease affects the antigenicity and bioactivity of TGF-ß2 in (CAGA)9-Luc CCL64 cells and in human hepatic cell lines via binding to TGF-ß type I receptor (TßRI). Tumor necrosis factor (TNF)-α facilitates this mechanism by increasing the colocalization of TßRI with NS3 protease on the surface of HCV-infected cells. An anti-NS3 antibody against computationally predicted binding sites for TßRI blocked the TGF-ß mimetic activities of NS3 in vitro and attenuated liver fibrosis in HCV-infected chimeric mice. These data suggest that HCV NS3 protease mimics TGF-ß2 and functions, at least in part, via directly binding to and activating TßRI, thereby enhancing liver fibrosis.

Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition.

Helix-loop-helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant-negative HLH (dnHLH) proteins lack DNA-binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho-physiological processes. We report the first structure of a free-standing human dnHLH protein, HHM (Human homologue of murine maternal Id-like molecule). HHM adopts a V-shaped conformation, with N-terminal and C-terminal five-helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N- and C-terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V-shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V-shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V-shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

Localization of Gts1p in cortical actin patches of yeast and its possible role in endocytosis.

Herein we report that Gts1p fused with green-fluorescent protein (GFP) is localized in the cortical actin patch besides nuclei in yeast and the cortical Gts1p changed its position together with the patch depending on the cell-cycle phase, while nuclear Gts1p accumulated predominantly in the budding phase. Whereas Gts1p does not directly bind to actin, it associated mainly with the actin-associated protein Pan1p. In the GTS1-deleted transformant gts1Delta, the number of cells containing either a fragmented vacuole or an enlarged single central vacuole increased and the uptake of the hydrophilic dye Lucifer yellow (LY) in the vacuole decreased. Further, gts1Delta transformed with a mutant Gts1p having two cysteine-to-alanine substitutions in a zinc finger resembling that of GTPase-activating proteins of ADP-ribosylation factors (ARF-GAP) neither recovered the LY uptake unlike gts1Delta transformed with the wild-type GTS1, nor reduced the average size of central vacuoles as much as the latter did. These results suggested that Gts1p in the actin patch is involved in the fluid-phase endocytosis and membrane trafficking for vacuole formation and that the putative ARF-GAP domain in Gts1p plays an important role in these functions.

The mechanism by which overexpression of Gts1p induces flocculation in a FLO8-inactive strain of the yeast Saccharomyces cerevisiae.

GTS1 induces flocculation when overexpressed in the Saccharomyces cerevisiae strain W303-1A, which carries a mutant FLO8, the activator of flocculin genes. Herein, we report that the GTS1-induced flocculation was flocculin-dependent in nature and was caused by expression of the major flocculin Flo1p. Gts1p bound to the repressor Sfl1p, and their interaction at the transcriptional level was shown by reporter gene assays using the FLO1 promoter, suggesting that Gts1p induces the expression of FLO1 by inhibiting Sfl1p. Furthermore, the Q-rich domain with the preceding 18 amino acids of Gts1p bound mediators for RNA polymerase II.

Gts1p stabilizes oscillations in energy metabolism by activating the transcription of TPS1 encoding trehalose-6-phosphate synthase 1 in the yeast Saccharomyces cerevisiae.

We reported previously that Gts1p regulates oscillations of heat resistance in concert with those of energy metabolism in continuous cultures of the yeast Saccharomyces cerevisiae by inducing fluctuations in the levels of trehalose, but not in those of Hsp104 (heat shock protein 104). Further, the expression of TPS1, encoding trehalose-6-phosphate synthase 1, and HSP104 was activated by Gts1p in combination with Snf1 kinase, a transcriptional activator of glucose-repressible genes, in batch cultures under derepressed conditions. Here we show that, in continuous cultures, the mRNA level of TPS1 increased 6-fold in the early respiro-fermentative phase, while that of HSP104 did not change. The expression of SUC2, a representative glucose-repressible gene encoding invertase, also fluctuated, suggesting the involvement of the Snf1 kinase in the periodic activation of these genes. However, this possibility was proven to be unlikely, since the oscillations in both TPS1 and SUC2 mRNA expression were reduced by approx. 3-fold during the transient oscillation in gts1Delta (GTS1-deleted) cells, in which the energy state determined by extracellular glucose and intracellular adenine nucleotide levels was comparable with that in wild-type cells. Furthermore, neither the mRNA level nor the phosphorylation status of Snf1p changed significantly during the oscillation. Thus we suggest that Gts1p plays a major role in the oscillatory expression of TPS1 and SUC2 in continuous cultures of Saccharomyces cerevisiae, and hypothesized that Gts1p stabilizes oscillations in energy metabolism by activating trehalose synthesis to facilitate glycolysis at the shift from the respiratory to the respiro-fermentative phase.

Gts1p activates SNF1-dependent derepression of HSP104 and TPS1 in the stationary phase of yeast growth.

We previously reported that the GTS1 product, Gts1p, plays an important role in the regulation of heat tolerance of yeast under glucose-limited conditions in either batch or continuous culture. Here we show that heat tolerance was decreased in GTS1-deleted and increased in GTS1-overexpressing cells under glucose-derepressed conditions during the batch culture and that the disruption of SNF1, a transcriptional activator of glucose-repressible genes, diminished this effect of GTS1. Intracellular levels of Hsp104 and trehalose, which were reportedly required for the acquisition of heat tolerance in the stationary phase of cell growth, were affected in both GTS1 mutants roughly in proportion to the gene dosage of GTS1, whereas those of other Hsps were less affected. The mRNA levels of genes for Hsp104 and trehalose-6-phosphate synthase 1 changed as a function of GTS1 gene dosage. The Q-rich domain of Gts1p fused with the DNA-binding domain of LexA activated the transcription of the reporter gene LacZ, and Gts1p lacking the Q-rich domain lost the activation activity of HSP104 and TPS1. Furthermore, Gts1p bound to subunits of Snf1 kinase, whereas it did not bind to DNA. Therefore, we suggested that GTS1 increases heat tolerance by mainly activating Snf1 kinase-dependent derepression of HSP104 and TPS1 in the stationary phase of yeast growth.