The structural basis for deubiquitination by the fingerless USP-type effector TssM.

Intracellular bacteria are threatened by ubiquitin-mediated autophagy, whenever the bacterial surface or enclosing membrane structures become targets of host ubiquitin ligases. As a countermeasure, many intracellular pathogens encode deubiquitinase (DUB) effectors to keep their surfaces free of ubiquitin. Most bacterial DUBs belong to the OTU or CE-clan families. The betaproteobacteria Burkholderia pseudomallei and Burkholderia mallei, causative agents of melioidosis and glanders, respectively, encode the TssM effector, the only known bacterial DUB belonging to the USP class. TssM is much shorter than typical eukaryotic USP enzymes and lacks the canonical ubiquitin-recognition region. By solving the crystal structures of isolated TssM and its complex with ubiquitin, we found that TssM lacks the entire "Fingers" subdomain of the USP fold. Instead, the TssM family has evolved the functionally analog "Littlefinger" loop, which is located towards the end of the USP domain and recognizes different ubiquitin interfaces than those used by USPs. The structures revealed the presence of an N-terminal immunoglobulin-fold domain, which is able to form a strand-exchange dimer and might mediate TssM localization to the bacterial surface.

Beyond the C-terminal Glycine of ATG8 Proteins - The Story of Some Neglected Amino Acids.

ATG8 proteins form a family of small ubiquitin-like modifiers, well-known for their importance in both macroautophagy and autophagy-independent processes. A unique feature of this protein family is their conjugation to membrane lipids through the covalent attachment of a glycine residue at the C-terminus of ATG8 proteins. Notably, most ATG8 proteins are expressed with additional amino acids at their C-terminus, shielding the key glycine residue. Consequently, lipidation requires the activation of the ATG8 precursors through proteolytic cleavage, known as priming. ATG4 proteases catalyze this priming process, and under physiological conditions, unprimed forms of ATG8 are not detected. This raises the question about the purpose of the C-terminal extension of ATG8 proteins. While the roles of lipidated and free, primed ATG8 proteins have been extensively studied, the potential function of their precursor form or the priming process itself remains largely unexplored. Here, we summarize information from existing literature and our own experiments to contribute to the understanding of these neglected amino acids.

Lane formation in gravitationally driven colloid mixtures consisting of up to three different particle sizes.

Brownian dynamics simulations are utilized to study segregation phenomena far from thermodynamic equilibrium. In the present study, we expand upon the analysis of binary colloid mixtures and introduce a third particle species to further our understanding of colloidal systems. Gravitationally driven, spherical colloids immersed in an implicit solvent are confined in two-dimensional linear microchannels. The interaction between the colloids is modeled by the Weeks-Chandler-Andersen potential, and the confinement of the colloids is realized by hard walls based on the solution of the Smoluchowski equation in half space. In binary and ternary colloidal systems, a difference in the driving force is achieved by differing colloid sizes but fixed mass density. We observe for both the binary and ternary systems that a driving force difference induces a nonequilibrium phase transition to lanes. For ternary systems, we study the tendency of lane formation to depend on the diameter of the medium-sized colloids. Here we find a sweet spot for lane formation in ternary systems. Furthermore, we study the interaction of two differently sized colloids at the channel walls. Recently we observed that driven large colloids push smaller colloids to the walls. This results in small particle lanes at the walls at early simulation times. In this work we additionally find that thin lanes are unstable and dissolve over very long time frames. Furthermore, we observe a connection between lane formation and the nonuniform distribution of particles along the channel length. This nonuniform distribution occurs either alongside lane formation or in shared lanes (i.e., lanes consisting of two colloid types).

N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling.

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

Cytoplasmic calcium influx mediated by plant MLKLs confers TNL-triggered immunity.

The plant homolog of vertebrate necroptosis inducer mixed-lineage kinase domain-like (MLKL) contributes to downstream steps in Toll-interleukin-1 receptor domain NLR (TNL)-receptor-triggered immunity. Here, we show that Arabidopsis MLKL1 (AtMLKL1) clusters into puncta at the plasma membrane upon TNL activation and that this sub-cellular reorganization is dependent on the TNL signal transducer, EDS1. We find that AtMLKLs confer TNL-triggered immunity in parallel with RPW8-type HeLo-domain-containing NLRs (RNLs) and that the AtMLKL N-terminal HeLo domain is indispensable for both immunity and clustering. We show that the AtMLKL HeLo domain mediates cytoplasmic Ca2+ ([Ca2+]cyt) influx in plant and human cells, and AtMLKLs are responsible for sustained [Ca2+]cyt influx during TNL-triggered, but not CNL-triggered, immunity. Our study reveals parallel immune signaling functions of plant MLKLs and RNLs as mediators of [Ca2+]cyt influx and a potentially common role of the HeLo domain fold in the Ca2+-signal relay of diverse organisms.

ERLIN1/2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway.

Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain-containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.