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Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and establish a safe, replicative lifestyle. The bacteria, once phagocytosed, reside in a vacuolar structure known as the Legionella-containing vacuole (LCV) within the host cells and rapidly subvert organelle trafficking events, block inflammatory responses, hijack the host ubiquitination system, and abolish apoptotic signaling. This arsenal of translocated effectors can manipulate the host factors in a multitude of different ways. These proteins also contribute to bacterial virulence by positively or negatively regulating the activity of one another. Such effector-effector interactions, direct and indirect, provide the delicate balance required to maintain cellular homeostasis while establishing itself within the host. This review summarizes the recent progress in our knowledge of the structure-function relationship and biochemical mechanisms of select effector pairs from Legionella that work in opposition to one another, while highlighting the diversity of biochemical means adopted by this intracellular pathogen to establish a replicative niche within host cells.

The ubiquitin (Ub)-proteasome system is the primary mechanism for maintaining protein homeostasis in eukaryotes, yet the underlying signaling events and specificities of its components are poorly understood. Proteins destined for degradation are tagged with covalently linked polymeric Ub chains and subsequently delivered to the proteasome, often with the assistance of shuttle proteins that contain Ub-like domains. This degradation pathway is riddled with apparent redundancy-in the form of numerous polyubiquitin chains of various lengths and distinct architectures, multiple shuttle proteins, and at least three proteasomal receptors. Moreover, the largest proteasomal receptor, Rpn1, contains one known binding site for polyubiquitin and shuttle proteins, although several studies have recently proposed the existence of an additional uncharacterized site. Here, using a combination of NMR spectroscopy, photocrosslinking, mass spectrometry, and mutagenesis, we show that Rpn1 does indeed contain another recognition site that exhibits affinities and binding preferences for polyubiquitin and Ub-like signals comparable to those of the known binding site in Rpn1. Surprisingly, this novel site is situated in the N-terminal section of Rpn1, a region previously surmised to be devoid of functionality. We identified a stretch of adjacent helices as the location of this previously uncharacterized binding site, whose spatial proximity and similar properties to the known binding site in Rpn1 suggest the possibility of multivalent signal recognition across the solvent-exposed surface of Rpn1. These findings offer new mechanistic insights into signal recognition processes that are at the core of the Ub-proteasome system.

Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K+ channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K+ channels, however, remain unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent nonglycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, nonglycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.

TRAF3 has diverse signaling functions, which vary by cell type. Uniquely in B lymphocytes, TRAF3 inhibits homeostatic survival. Highlighting the role of TRAF3 as a tumor suppressor, loss-of-function TRAF3 mutations are associated with human B-cell malignancies, while B-cell-specific deletion of TRAF3 in mice leads to autoimmunity and lymphoma development. The role of TRAF3 in inhibiting noncanonical NF-κB activation, CD40 and BAFF-R signaling to B cells is well documented. In contrast, TRAF3 enhances many T-cell effector functions, through associating with and enhancing signaling by the T-cell receptor (TCR)-CD28 complex. The present study was designed to determine the role of TRAF3 in signaling via the B-cell antigen receptor (BCR). The BCR is crucial for antigen recognition, survival, proliferation, and antibody production, and defects in BCR signaling can promote abnormal survival of malignant B cells. Here, we show that TRAF3 is associated with both CD79B and the BCR-activated kinases Syk and Btk following BCR stimulation. BCR-induced phosphorylation of Syk and additional downstream kinases was increased in TRAF3−/− B cells, with regulation observed in both follicular and marginal zone B-cell subsets. BCR stimulation of TRAF3−/− B cells resulted in increased surface expression of MHC-II, CD80, and CD86 molecules. Interestingly, increased survival of TRAF3−/− primary B cells was resistant to inhibition of Btk, while TRAF3-deficient malignant B-cell lines showed enhanced sensitivity. TRAF3 serves to restrain normal and malignant BCR signaling, with important implications for its role in normal B-cell biology and abnormal survival of malignant B cells.

The voltage-gated potassium channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA, 10 nM) diminished Kv1.5 current (IKv1.5) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 channel function under PKC activation conditions.