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Cereal products like flour and bread are known to trigger diseases such as wheat allergy, celiac disease and non-celiac wheat sensitivity (NCWS). Some of these diseases are caused by allergenic proteins, the expression of which might vary depending on the grain type and manufacturing processes. Therefore, we examined the protein composition and abundance of potentially allergenic proteins in flours from bread wheat, spelt and rye, and corresponding breads.Using Nano-LC-ESI-MS/MS and label free quantification (LFQ) we analyzed the proteome of six different bread flours (wholegrain and superfine flours from rye, spelt and bread wheat) and 14 bread types (yeast and sourdough fermented breads from all flours and wheat breads plus/minus bread improver). Potentially allergenic proteins in flours and breads were functionally categorized using the Pfam database and relatively quantified by LFQ.We could show that almost equal numbers of proteins can be identified in rye- and spelt samples compared to wheat samples using the Uniprot bread wheat protein database, indicating high sequence conservation between cereals. In total, 4424 proteins were identified in the 20 flour and bread samples. The average number of identified proteins in flour (2719 ± 243) was slightly higher than in bread (2283 ± 232; P 0.001). In wheat- and spelt wholegrain flour higher protein numbers (wheat: 2891 ± 90; spelt: 2743 ± 140) were identified on average than in superfine flour (wheat: 2562 ± 79; P = 0.009; spelt: 2431 ± 140; P = 0.004). Neither the absolute number nor the abundance distribution of potentially allergenic proteins were dependent on the flour type or the fermentation process, but known allergenic proteins like gliadins showed higher relative abundance in spelt- and wheat samples, compared to rye samples.We provide comprehensive proteome data for six flour types and related breads showing that the grain species have greater influence on proteome composition than milling and fermentation processes. Our data indicate that allergenic proteins are not selectively degraded during bread production and are more abundant in bread wheat and spelt compared to rye.Our proteomics study revealed that bread contains a number of potentially and proven allergenic proteins. Most likely allergenicity is not dependent on milling or conventional fermentation processes, but on the grain type. Relative abundance of allergenic proteins was higher in spelt- and wheat samples than in rye samples. Considering rye bread as better suited to atopic individuals predisposed to react to cereal allergens, clinical trials are warranted to verify this assumption.

Viruses that generate capped RNA lacking 2′O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2′O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2′O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2′O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2′O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells., Author Summary Cellular messenger RNAs of higher eukaryotes are capped with a methylated guanine and, in addition, methylated at the 2′O position of the first ribose. Viruses unable to methylate their RNA at the 2′O position of the cap and viruses generating uncapped RNA with 5′ triphosphate groups are inhibited by an antiviral complex of different IFIT proteins. How IFIT proteins restrict viruses lacking 2′O methylation at the RNA cap remained unclear. We used a mass spectrometry-based approach to identify proteins binding to capped RNA with different methylation states. We found that IFIT1 directly binds to capped RNA and that this binding was dependent on the methylation state of the cap. Having identified IFIT1 as being central for recognition of 2′O-unmethylated viral RNA we further examined the mode of action of IFITs in vitro and in vivo. Our experiments clearly show that the antiviral mechanism of IFIT1 is based on sequestration of viral RNA lacking cap 2′O methylation, thereby selectively preventing translation of viral RNA. Our data establish IFIT1 as a general sensor for RNA 5′ end structures and provide an important missing link in our understanding of the antiviral activity of IFIT proteins.

HIV-1 Vpu induces downregulation of cell surface NTB-A to evade lysis of HIV-1 infected cells by NK cells. Here we show that Vpu affects the anterograde transport and the glycosylation pattern of NTB-A by a mechanism that is distinct from the Vpu induced downregulation of CD4 and tetherin. In the presence of Vpu, only the high mannose form of NTB-A was detectable, suggesting that Vpu prevented the formation of the mature form of NTB-A. This phenomenon is associated with the ability of Vpu to downregulate cell surface NTB-A by retention of NTB-A within the Golgi-compartment. Furthermore, the Vpu-mediated effect on NTB-A glycosylation is highly conserved among Vpu proteins derived from HIV-1 and SIV and corresponds to the level of downregulation of NTB-A. Together, these results suggest that the reduction of NTB-A from the cell surface is associated with the Vpu-mediated effect on the glycosylation pattern of newly synthesized NTB-A molecules.

The Pyrin and HIN200 domain-containing (PYHIN) protein family consists of AIM2, IFI16, MNDA, PYHIN1 and POP3 in humans and at least 13 proteins in mouse. Mouse AIM2 and p204 have been previously described to be intracellular dsDNA sensors leading to caspase-1 activation and STING-dependent IFN- β induction respectively. However the function of most mouse PYHIN proteins remains unknown. Here we characterized one such mouse PYHIN protein, p207. P207 contains an N-terminal Pyrin and a C-terminal HINB domain that share strong sequence similarity with the corresponding domains of p204 (85% and 92% respectively). P207 is highly expressed in BMDMs and BMDCs and its expression is further enhanced after dsDNA stimulation of cells in an IFN-dependent manner. In contrast to p204, reduction of p207 expression by shRNA had no effect on DNA-induced IFN- β . Rather, p207 shRNA studies in macrophage cell lines showed an impairment in TNF mRNA induction following stimulation with dsDNA, transfected poly(I:C) or LPS as well as after infection with HSV. Induction of other LPS-dependent genes including CCL5, IL-1, IL-12 or IL-6 was not p207-dependent. Further, ectopic expression of p207 induced a murine TNF promoter reporter, while p207 shRNA inhibited LPS-stimulated RNA polymerase II recruitment to the TNF promoter. Furthermore, p207 interactome analysis by mass spectrometry revealed a list of potential interaction partners involved in transcription. These candidates and their involvement in p207-mediated effects on TNF induction are being currently pursued. These results demonstrate the existence of a novel PYHIN-dependent mechanism of regulation of TNF induction during pattern recognition receptor responses. Thus mouse p207 is another PYHIN protein involved in mediating innate immune responses. Further, the requirement for a PYHIN protein for maximal TNF responses may be conserved in humans.