Development of microwave-assisted methods to aid in carbohydrate analysis

This study involved development of three high-throughput chemical techniques to assist in carbohydrate analysis. The methods utilized both microwave radiation and a volatile organic base to achieve short reaction times and high product yield. Two methods, one reductive and one non-reductive, released intact O-glycans for structural analysis, and one method aided in determination of the site of O-glycosylation on the peptide. Optimal reaction conditions for each method were determined on standard glycoproteins. In comparison to common release methods, microwave-assisted reductive O-glycan release provided higher yields of O-glycans in every case. O-glycans were released rapidly and were able to be subsequently labeled for chromatographic separations by the non-reductive release method. The O-glycosylation site was determined following release by the formation of peptide-dimethylamine adducts, which, coupled with the addition of a deuterium label further aided in selection of glycopeptides for analysis

The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1-Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. To map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly but, rather, binds both the binary Sec9-Sso1 and ternary Sec9-Sso1-Snc2 SNARE complexes. Together, these results suggest a new model in which Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions

FANCJ/BACH1 Acetylation at Lysine 1249 Regulates the DNA Damage Response

BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance

Serine/threonine acetylation of TGFbeta-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling

The Gram-negative bacteria Yersinia pestis, causative agent of plague, is extremely virulent. One mechanism contributing to Y. pestis virulence is the presence of a type-three secretion system, which injects effector proteins, Yops, directly into immune cells of the infected host. One of these Yop proteins, YopJ, is proapoptotic and inhibits mammalian NF-kappaB and MAP-kinase signal transduction pathways. Although the molecular mechanism remained elusive for some time, recent work has shown that YopJ acts as a serine/threonine acetyl-transferase targeting MAP2 kinases. Using Drosophila as a model system, we find that YopJ inhibits one innate immune NF-kappaB signaling pathway (IMD) but not the other (Toll). In fact, we show YopJ mediated serine/threonine acetylation and inhibition of dTAK1, the critical MAP3 kinase in the IMD pathway. Acetylation of critical serine/threonine residues in the activation loop of Drosophila TAK1 blocks phosphorylation of the protein and subsequent kinase activation. In addition, studies in mammalian cells show similar modification and inhibition of hTAK1. These data present evidence that TAK1 is a target for YopJ-mediated inhibition

Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae

The budding yeast heat shock protein Hsp42 coaggregates with misfolded proteins and may link those aggregates to further sorting factors

FANCJ^K1249R or FANCJ^K1249Q promotes polη- or Rad54-dependent repair, respectively.

<p>A. FA-J cells expressing acetylation mutants have a distinct response and reliance on repair or tolerance factors for DNA damage survival. The FA-J cell lines were transfected with siRNA against Luc, Polη, or Rad54. The cells were treated with indicated doses of zeocin, UV, or as in B., with MMC and the percent survival was calculated 8 days later. Data represent mean percent ± s.d. of survival from three independent experiments. C. Cells were collected and analyzed for expression with the indicated antibodies.</p

FANCJ acetylation is induced after DNA damage.

<p>A. Endogenous FANCJ is acetylated in response DNA damage. MCF7 and HeLa cells were left untreated (UT) or treated with zeocin (6.25 µg/ml for 1 h), MMC (250 nM for 1 h), UV (30 J/m²), MMS (300 µg/ml for 4 h), HU (1 mM for 24 h), or CPT (1 µM for 1 h). Cell lysates were collected at distinct times post damage (zeocin 24 h, MMC 24 h or as indicated, UV 6 h, CPT 24 h, MMS 4 h, and HU 24 h) and analyzed for expression and/or acetylation following immunoprecipitation with the indicated antibodies. B. Exogenous FANCJ is acetylated on lysine 1249 in response to DNA damage. Myc-tagged FANCJ wild-type or mutant species and CBP were co-transfected into 293T cells and left untreated (UT) or treated with zeocin (12.5 µg/ml for 1 h or C. CPT (1 µM for 1 h). Cells were processed at different time points post DNA damage and analyzed for expression and/or acetylation following immunoprecipitation with the indicated antibodies.</p

FANCJ acetylation mutants are functional.

<p>A. The acetylation mutants are expressed in FA-J cells. FA-J cells were complemented with vector, FANCJ^WT, FANCJ^K1249R, or FANCJ^K1249Q. The FA-J cell lines were collected and analyzed or B. lysates were immunoprecipitated with FANCJ antibodies and immunoblot was performed with the indicated antibodies. C. The acetylation mutants localize in nuclear foci of FA-J cells. The FA-J cell lines were seeded onto 6-well plates and incubated overnight. The cells were treated with 1 mM HU and 24 h later immunoflourescence was performed with the indicated antibodies. D. The FA-J cell lines have similar cell cycle profiles. The FA-J cells lines were collected and analyzed by FACS to determine the percentage of cells with 2N and 4N DNA content. E. Expression of acetylation mutants restores MMC resistance. The FA-J cell lines were seed onto 6 well plates and incubated overnight. The cells were either left untreated or treated with increasing doses of MMC. Cells were counted 8 days later and percent survival was calculated. Data represent mean percent ± s.d. of survival from three independent experiments. F. Expression of acetylation mutants restores G2/M checkpoint exit. The FA-J cell lines were untreated or treated with 0.25 µg/ml melphalan, collected at the indicated times, and analyzed by FACS to determine the percentage of cells in G2/M. Data represent mean percent ± s.d. of survival from three independent experiments.</p

FANCJ and its acetylation at 1249 promote an RPA response.

<p>A. Deficiency in FANCJ or its acetylation impairs the CPT-induced RPA focus formation. The FA-J cell lines were seeded onto 6-well plates, incubated overnight, left untreated or treated with CPT 1 h and immunoflourescence was performed with the indicated antibodies. The percent of cells with RPA foci was quantified and graphed. Data represent mean ± s.d. from three independent experiments. B. FANCJ and its acetylation promote RPA phosphorylation at 1 h post-CPT. The complemented FA-J cell lines were either left untreated or treated for 1 h with the indicated dose of CPT and analyzed 1 h post-treatment. Cell lysates were collected, lysed, and analyzed with the indicated antibodies. C. FANCJ acetylation promotes RPA phosphorylation at times greater than 1 h post-CPT. Same as above but collected at the time points indicated.</p