Far Upstream Binding Protein 1 (FUBP1) participates in translational regulation of Nrf2 protein under oxidative stress.

Oxidative stress is ubiquitously involved in disease etiology or progression. While the damaging effects have been well characterized, how cells deal with oxidative stress for prevention or removal of damage remains to be fully elucidated. Works from our laboratory have revealed de novo Nrf2 protein translation when cells are encountering low to mild levels of oxidative stress. Nrf2 encodes a transcription factor controlling a myriad of genes important for antioxidation, detoxification, wound repair and tissue remodeling. Here we report a role of FUBP1 in regulating de novo Nrf2 protein translation. An increase of FUBP1 binding to Nrf2 5'UTR due to H2O2 treatment has been found by LC-MS/MS, Far Western blot and ribonucleoprotein immunoprecipitation assays. Blocking FUBP1 expression using siRNA abolished H2O2 from inducing Nrf2 protein elevation or Nrf2 5'UTR activity. While no nuclear to cytoplasmic translocation was detected, cytosolic redistribution to the ribosomal fractions was observed due to oxidant treatment. The presence of FUBP1 in 40/43S ribosomal fractions confirm its involvement in translation initiation of Nrf2 protein. When tested by co-immunoprecipitation with eIF4E, eIF2a, eIF3η and eIF1, only eIF3η was found to gain physical interaction with FUBP1 due to H2O2 treatment. Our data support a role of FUBP1 for promoting the attachment of 40S ribosomal subunit to Nrf2 mRNA and formation of 43S pre-initiation complex for translation initiation of Nrf2 protein under oxidative stress.

Histone deacetylase 6 associates with ribosomes and regulates de novo protein translation during arsenite stress.

Histone deacetylase 6 (HDAC6) is known as a cytoplasmic enzyme that regulates cell migration, cell adhesion, and degradation of misfolded proteins by deacetylating substrates such as α-tubulin and Hsp90. When HaCaT keratinocytes were exposed to 1-200µM sodium arsenite, we observed perinuclear localization of HDAC6 within 30 min. Although the overall level of HDAC6 protein did not change, sodium arsenite caused an increase of HDAC6 in ribosomal fractions. Separation of ribosomal subunits versus intact ribosomes or polysomes indicated that HDAC6 was mainly detected in 40/43S fractions containing the small ribosomal subunit in untreated cells but was associated with 40/43S and 60/80S ribosomal fractions in arsenite-treated cells. Immunocytochemistry studies revealed that arsenite caused colocalization of HDAC6 with the ribosomal large and small subunit protein L36a and S6. Both L36a and S6 were detected in the immunocomplex of HDAC6 isolated from arsenite-treated cells. The observed physical interaction of HDAC6 with ribosomes pointed to a role of HDAC6 in stress-induced protein translation. Among arsenite stress-induced proteins, de novo Nrf2 protein translation was inhibited by Tubastatin A. These data demonstrate that HDAC6 was recruited to ribosomes, physically interacted with ribosomal proteins, and regulated de novo protein translation in keratinocytes responding to arsenite stress.

A naturally occurring Rgg variant in serotype M3 Streptococcus pyogenes does not activate speB expression due to altered specificity of DNA binding.

The transcriptional regulator Rgg of Streptococcus pyogenes is essential for expression of the secreted cysteine protease SpeB. Although all isolates of S. pyogenes possess the speB gene, not all of them produce the protein in vitro. In a murine model of infection, the absence of SpeB production is associated with invasive disease. We speculated that naturally occurring mutations in rgg, which would also abrogate SpeB production, may be present in invasive isolates of S. pyogenes. Examination of the inferred Rgg sequences available in public databases revealed that the rgg gene in strain MGAS315 (a serotype M3 strain associated with invasive disease) encodes a proline at amino acid position 103 (Rgg(103P)); in contrast, all other strains encode a serine at this position (Rgg(103S)). A caseinolytic assay and Western blotting indicated that strain MGAS315 does not produce SpeB in vitro. Gene-swapping experiments showed that the rgg gene of MGAS315 is solely responsible for the lack of SpeB expression. In contrast to Rgg(103S), Rgg(103P) does not bind to the speB promoter in gel shift assays, which correlates with a lack of speB expression. Despite its inability to activate speB expression, Rgg(103P) retains the ability to bind to DNA upstream of norA and to influence its expression. Overall, this study illustrates how variation at the rgg locus may contribute to the phenotypic diversity of S. pyogenes.

The Rgg regulator of Streptococcus pyogenes influences utilization of nonglucose carbohydrates, prophage induction, and expression of the NAD-glycohydrolase virulence operon.

The expression of many virulence-associated genes in Streptococcus pyogenes is controlled in a growth phase-dependent manner. Unlike the model organisms Escherichia coli and Bacillus subtilis, such regulation is apparently not dependent upon alternative sigma factors but appears to rely on complex interactions among several transcriptional regulators, including Rgg. The purpose of this study was to identify changes in gene expression associated with inactivation of the rgg gene in S. pyogenes strain NZ131 (serotype M49). To this end, the transcriptomes of wild-type and rgg mutant strains were analyzed during both the exponential and postexponential phases of growth using Affymetrix NimbleExpress gene chips. Genomewide differences in transcript levels were identified in both phases of growth. Inactivation of rgg disrupted coordinate expression of genes associated with the metabolism of nonglucose carbon sources, such as fructose, mannose, and sucrose. The changes were associated with an inability of the mutant strain to grow using these compounds as the primary carbon source. Bacteriophage transcript levels were also altered in the mutant strain and were associated with decreased induction of at least one prophage. Finally, transcripts encoding virulence factors involved in cytolysin-mediated translocation of NAD-glycohydrolase, including the immunity factor IFS and the cytolysin (streptolysin O [SLO]), were more abundant in the mutant strain, which correlated with the amount of NADase and SLO activities in culture supernatant fluids. The results provide further evidence that Rgg contributes to growth phase-dependent gene regulation in strain NZ131.