Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium expresses two type III secretion systems, T3SS1 and T3SS2, which are encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. These are essential virulent factors that secrete more than 40 effectors that are translocated into host animal cells. This study focuses on three of these effectors, SlrP, SspH1, and SspH2, which are members of the NEL family of E3 ubiquitin ligases. We compared their expression, regulation, and translocation patterns, their role in cell invasion and intracellular proliferation, their ability to interact and ubiquitinate specific host partners, and their effect on cytokine secretion. We found that transcription of the three genes encoding these effectors depends on the virulence regulator PhoP. Although the three effectors have the potential to be secreted through T3SS1 and T3SS2, the secretion of SspH1 and SspH2 is largely restricted to T3SS2 due to their expression pattern. We detected a role for these effectors in proliferation inside fibroblasts that is masked by redundancy. The generation of chimeric proteins allowed us to demonstrate that the N-terminal part of these proteins, containing the leucine-rich repeat motifs, confers specificity towards ubiquitination targets. Furthermore, the polyubiquitination patterns generated were different for each effector, with Lys48 linkages being predominant for SspH1 and SspH2. Finally, our experiments support an anti-inflammatory role for SspH1 and SspH2.

SNRPD2 Is a Novel Substrate for the Ubiquitin Ligase Activity of the Salmonella Type III Secretion Effector SlrP.

SlrP is a protein with E3 ubiquitin ligase activity that is translocated by Salmonella enterica serovar Typhimurium into eukaryotic host cells through a type III secretion system. A yeast two-hybrid screen was performed to find new human partners for this protein. Among the interacting proteins identified by this screen was SNRPD2, a core component of the spliceosome. In vitro ubiquitination assays demonstrated that SNRPD2 is a substrate for the catalytic activity of SlrP, but not for other members of the NEL family of E3 ubiquitin ligases, SspH1 and SspH2. The lysine residues modified by this activity were identified by mass spectrometry. The identification of a new ubiquitination target for SlrP is a relevant contribution to the understanding of the role of this Salmonella effector.

The NEL Family of Bacterial E3 Ubiquitin Ligases.

Some pathogenic or symbiotic Gram-negative bacteria can manipulate the ubiquitination system of the eukaryotic host cell using a variety of strategies. Members of the genera Salmonella, Shigella, Sinorhizobium, and Ralstonia, among others, express E3 ubiquitin ligases that belong to the NEL family. These bacteria use type III secretion systems to translocate these proteins into host cells, where they will find their targets. In this review, we first introduce type III secretion systems and the ubiquitination process and consider the various ways bacteria use to alter the ubiquitin ligation machinery. We then focus on the members of the NEL family, their expression, translocation, and subcellular localization in the host cell, and we review what is known about the structure of these proteins, their function in virulence or symbiosis, and their specific targets.

Non-productive angiogenesis disassembles Aß plaque-associated blood vessels.

The human Alzheimer's disease (AD) brain accumulates angiogenic markers but paradoxically, the cerebral microvasculature is reduced around Aß plaques. Here we demonstrate that angiogenesis is started near Aß plaques in both AD mouse models and human AD samples. However, endothelial cells express the molecular signature of non-productive angiogenesis (NPA) and accumulate, around Aß plaques, a tip cell marker and IB4 reactive vascular anomalies with reduced NOTCH activity. Notably, NPA induction by endothelial loss of presenilin, whose mutations cause familial AD and which activity has been shown to decrease with age, produced a similar vascular phenotype in the absence of Aß pathology. We also show that Aß plaque-associated NPA locally disassembles blood vessels, leaving behind vascular scars, and that microglial phagocytosis contributes to the local loss of endothelial cells. These results define the role of NPA and microglia in local blood vessel disassembly and highlight the vascular component of presenilin loss of function in AD.