Members of the Legionella pneumophila Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination.

Legionella pneumophila establishes a replication vacuole by translocating hundreds of protein effectors through a type IV secretion system (T4SS). Among these translocated effectors are members of the Sde family, which catalyze phosphoribosyl-linked ubiquitination (pR-Ub) of host targets. Previous work has posited that Sde proteins solely target serine (Ser) residues within acceptor protein substrates. We show here that SdeC-mediated pR-Ub modification results from a stepwise reaction that also modifies tyrosine (Tyr) residues. Unexpectedly, the presence of an HA tag on Ub resulted in poly-pR-ubiquitination, consistent with the HA tag acting as an acceptor target. Interrogation of phosphoribosyl-linked HA-Ub revealed that Tyr4 was the preferred targeted residue, based on LC-MS/MS analysis of the crosslinked product. Further analysis using synthetic HA variants revealed promiscuous modification of Tyr, as crosslinking was prevented only by constructing a triple mutant in which all three Tyr within the HA sequence were substituted with Phe. Although previous work has indicated that Ser is the sole acceptor residue, we found no evidence of Ser preference over Tyr using Tyr → Ser replacement mutants. This work demonstrates that pR-ubiquitination by the Sde family is not limited to Ser-modification as previously proposed, and broadens the potential sites targeted by this family.

A Systematic Study of Selective Protein Glycation.

Glycation is a non-enzymatic post-translational modification (PTM) that remains poorly understood, largely because it is unknown how it occurs selectively. Using mass spectrometry, it was possible to evaluate total glycation levels, identify distinct glycated products, assign unique glycation sites, and correlate these data with chemical and structural features for a panel of proteins glycated in vitro. It was determined that the extent of glycation does not correlate with pKa or surface exposure at reactive sites. Rather, the data reveal that primary sequence dictates the overall likelihood that a site will become glycated, while surrounding structure further sculpts the glycation outcome. Clustered acidic residues were found to prevent glycation, whereas a combination of tyrosine and polar residues appear to promote glycation. This work contributes important new knowledge about the molecular features that govern selective glycation.

A Single Legionella Effector Catalyzes a Multistep Ubiquitination Pathway to Rearrange Tubular Endoplasmic Reticulum for Replication.

Intracellular pathogens manipulate host organelles to support replication within cells. For Legionella pneumophila, the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated replication compartment. We show here that the bacterial Sde proteins target host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule rearrangements as well as alterations in Rtn4 associated with the replication compartment. These rearrangements are triggered via Sde-promoted ubiquitin transfer to Rtn4, occurring almost immediately after bacterial uptake. Ubiquitin transfer requires two sequential enzymatic activities from a single Sde polypeptide: an ADP-ribosyltransferase and a nucleotidase/phosphohydrolase. The ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase, resulting in either transfer of ubiquitin to Rtn4 or phosphoribosylation of ubiquitin in the absence of a ubiquitination target. Therefore, a single bacterial protein drives a multistep biochemical pathway to control ubiquitination and tubular ER function independently of the host ubiquitin machinery.

Teen responses when a younger school-age sibling has been bullied.

The prevalence of bullying among children, and the sometimes tragic consequences as a result, has become a major concern in schools. The larger research for this study reported on in-depth interviews with 28 elementary and middle school-age boys and girls (7-12 years) who had experienced various forms of bullying and relational aggression by their peers, mostly on school grounds, and the responses of their parents and teachers. Responses of the children's teen siblings to the younger child's revelations of being bullied are the focus of this report. In-depth interviews with each teen sibling (n = 28) and with each bullied child revealed how the children viewed the teen siblings' supportive strategies. Almost all the children (89%) reported that their older siblings talked with them and offered advice. The teen siblings shared with the younger ones that they too (71%) had been bullied, or they knew someone who had been bullied (18%). Teens gave the advice to 'bully back' to 11% and advice to 'tell someone' to 32% of the younger children. The children felt quite positive about their older siblings' advice (89%), which did differ depending on the bullied child's gender. Teen siblings gave advice to 'avoid bullies' to 77% of female and to 27% of male younger children.