The SidE family of Legionella pneumophila effectors is a unique group of ubiquitin-modifying enzymes. Along with catalyzing NAD+-dependent ubiquitination of certain host proteins independent of the canonical E1/E2/E3 pathway, they have also been shown to produce phosphoribosylated free ubiquitin. This modified ubiquitin product is incompatible with conventional E1/E2/E3 ubiquitination processes, with the potential to lock down various cellular functions that are dependent on ubiquitin signaling. Here, we show that in addition to free ubiquitin, Lys63-, Lys48-, Lys11-, and Met1-linked diubiquitin chains are also modified by SdeA in a similar fashion. Both the proximal and distal ubiquitin moieties are targeted in the phosphoribosylation reaction. Furthermore, this renders the ubiquitin chains unable to be processed by a variety of deubiquitinating enzymes. These observations broaden the scope of SdeA’s modulatory functions during Legionella infection.

Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-minute gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5 μm in diameter, 2) superficially porous particles of 3 μm in diameter with a shell of 0.2 μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.

In a radical departure from the classical E1−E2−E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD + to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD + analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.

Currently, SiGe-B epitaxy is a leading technology to induce strain in PMOS channel and improve the hole mobility to achieve better device performance [1-2]. In practice, we observe that the device performance strongly depends on the dopant concentration, especially boron concentration. It is shown that the Acoustic Control System [ACS] is able to actively respond to instantaneous variations of incoming gas and achieve better dopant control [3]. In this paper, we report reduction of boron concentration variation using ACS technology for SiGe-B epitaxy.

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SiGe-B source/drain (S/D) epitaxy is successfully used in mass production of high performance CMOS device (1-2). Due to the difference in free energy across the SiGe-B/substrate interface, there is boron segregation at the interface (3-4). In addition, low level of contamination also contributes to the pile-up of boron at the interface (5). In PMOS device manufacturing, the excessive boron will diffuse into the channel region in the high temperature processes after EPI S/D, which adversely affects device performance (5). In this paper, influence of temperature on boron concentration in the bulk and at the interface is reported.