Molecular and Biological Characterization of Streptococcal SpyA-mediated ADP-ribosylation of Intermediate Filament Protein Vimentin

Abstract

Background: SpyA is a streptoccocal ADP-ribosyltransferase that modifies vimentin. Results: Vimentin is modified in the regulatory N-terminal head domain, and treatment with SpyA results in a defect in filamentation. Conclusion: Vimentin is a target substrate of SpyA, and ADP-ribosylation affects polymerization dynamics. Significance: Vimentin filaments are disrupted by a bacterial ADP-ribosyltransferase, SpyA, and ADP-ribosylation of vimentin regulates filament formation.

Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells*

“…Subsequently, cells were washed three times with 1ϫPBS thoroughly and examined using a LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). Whole cells were photographed with the image focused on the vimentin fibers localized closest to the cell periphery as described previously (33,34) using an OMX V4™ Superresolution Imaging Systems (GE Healthcare). All analyses and quantifications were performed in a blinded manner.…”

A SIRT4-like auto ADP-ribosyltransferase is essential for the environmental growth of <italic>Mycobacterium smegmatis</italic>

“…Two micrograms of MSMEG_4620 was incubated with 50 mM PIPES or Tris-HCl ( pH 7.2), 150 mM NaCl, and 50 µM biotin-NAD at 25°C for 3 h. After that, the reaction was terminated by boiling for 5 min, and samples were incubated with 0.5 M NaCl, 10 mM HgCl 2 , or 2 M NH 2 OH for additional 2 h at 30°C [13,23]. Finally, samples were subject to western blot analysis as described above.…”

“…To detect MSMEG_4620 ADP-ribosylation activity, 50 µM biotinylated NAD (6-biotin-17-NAD; R&D, Minneapolis, USA) was incubated with 50 mM Tris-HCl ( pH 7.2), 150 mM NaCl and 2 µg MSMEG_4620 at 25°C for 3 h [13]. The samples were prepared and resolved on sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membrane.…”

“…Mono-ADP-ribosylation often occurs on arginine, cysteine, glutamine, or lysine residues, which can be differentiated by chemical stability assay. For example, NH 2 OH can specifically break the ADP-ribosyl-arginine linkage other than linkages with other residues [13][14][15][16]. In fact, two kinds of ADP-ribosylation have been reported in prokaryotes.…”

“…In response to a negative stimulus such as the presence of ammonium or a decrease in the available cell energy, DraT can ADP-ribosylate Fe protein which in turn dissociates from MoFe protein and ultimately inhibits the nitrogenase activity, while DraG can remove the ADP-ribose moiety from Fe protein and therefore reactivate the nitrogenase [17,18]. In addition to DraT, some toxins secreted by pathogens also possess ADP-ribosylation activity and can disrupt the essential functions of the host cells [13,16,19]. For example, cholera toxin and pertussis toxin can ADP-ribosylate regulatory G proteins and then interfere with their signal transduction.…”