A functional screen for ubiquitin regulation identifies an E3 ligase secreted by .

Ubiquitin signaling controls many aspects of eukaryotic biology, including targeted protein degradation and immune defense. Remarkably, invading bacterial pathogens have adapted secreted effector proteins that hijack host ubiquitination to gain control over host responses. These ubiquitin-targeted effectors can exhibit, for example, E3 ligase or deubiquitinase activities, often without any sequence or structural homology to eukaryotic ubiquitin regulators.

Ubiquitylation: Sword and shield in the bacterial arsenal.

In two recent studies in Nature, Hör et al. and Chambers et al. report that ubiquitin-like conjugation in bacteria antagonizes phage replication.

Discovery of ester-linked ubiquitylation of PARP10 mono-ADP-ribosylation in cells: a dual post-translational modification on Glu/Asp side chains.

The prevailing view on post-translational modifications (PTMs) is that amino acid side chains in proteins are modified with a single PTM at any given time. However, a growing body of work has demonstrated crosstalk between different PTMs, some occurring on the same residue. Such interplay is seen with ADP-ribosylation and ubiquitylation, where specialized E3 ligases ubiquitylate targets for proteasomal degradation in an ADP-ribosylation-dependent manner.

Bacterial esterases reverse lipopolysaccharide ubiquitylation to block host immunity.

Aspects of how Burkholderia escape the host's intrinsic immune response to replicate in the cell cytosol remain enigmatic. Here, we show that Burkholderia has evolved two mechanisms to block the activity of Ring finger protein 213 (RNF213)-mediated non-canonical ubiquitylation of bacterial lipopolysaccharide (LPS), thereby preventing the initiation of antibacterial autophagy. First, Burkholderia's polysaccharide capsule blocks RNF213 association with bacteria and second, the Burkholderia deubiquitylase (DUB), TssM, directly reverses the activity of RNF213 through a previously unrecognized esterase activity.

The acetylase activity of Cdu1 regulates bacterial exit from infected cells by protecting effectors from degradation.

Many cellular processes are regulated by ubiquitin-mediated proteasomal degradation. Pathogens can regulate eukaryotic proteolysis through the delivery of proteins with de-ubiquitinating (DUB) activities. The obligate intracellular pathogen secretes Cdu1 (ChlaDUB1), a dual deubiquitinase and Lys-acetyltransferase, that promotes Golgi remodeling and survival of infected host cells presumably by regulating the ubiquitination of host and bacterial proteins.

Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here we show that its lysosomal degradation is dependent on ubiquitylation at Lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes.

Bacterial ligases reveal fundamental principles of polyubiquitin specificity.

Homologous to E6AP C terminus (HECT) E3 ubiquitin (Ub) ligases direct substrates toward distinct cellular fates dictated by the specific form of monomeric or polymeric Ub (polyUb) signal attached. How polyUb specificity is achieved has been a long-standing mystery, despite extensive study in various hosts, ranging from yeast to human. The bacterial pathogens enterohemorrhagic Escherichia coli and Salmonella Typhimurium encode outlying examples of "HECT-like" (bHECT) E3 ligases, but commonalities to eukaryotic HECT (eHECT) mechanism and specificity had not been explored.

Allelic strengths of encephalopathy-associated variants correlate between in vivo and in vitro assays.

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and endoplasmic reticulum stress. Variants in the gene are associated with developmental and epileptic encephalopathy 44 (DEE44), an autosomal recessive disorder characterized by early-onset encephalopathy, movement abnormalities, global developmental delay, intellectual disability, and seizures. DEE44 is caused by at least 12 different missense variants described as loss of function (LoF), but the relationships between genotypes and molecular or clinical phenotypes remain to be established.

Ubiquitin-targeted bacterial effectors: rule breakers of the ubiquitin system.

Regulation through post-translational ubiquitin signaling underlies a large portion of eukaryotic biology. This has not gone unnoticed by invading pathogens, many of which have evolved mechanisms to manipulate or subvert the host ubiquitin system. Bacteria are particularly adept at this and rely heavily upon ubiquitin-targeted virulence factors for invasion and replication.

Dedicated bacterial esterases reverse lipopolysaccharide ubiquitylation to block immune sensing.

Pathogenic bacteria have evolved diverse mechanisms to counteract cell-autonomous immunity, which otherwise guards both immune and non-immune cells from the onset of an infection. The versatile immunity protein Ring finger protein 213 (RNF213) mediates the non-canonical ester-linked ubiquitylation of lipopolysaccharide (LPS), marking bacteria that sporadically enter the cytosol for destruction by antibacterial autophagy. However, whether cytosol-adapted pathogens are ubiquitylated on their LPS and whether they escape RNF213-mediated immunity, remains unknown.

Allelic strengths of encephalopathy-associated variants correlate between and assays.

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and ER stress. Variants in the gene are associated with developmental and epileptic encephalopathy 44 (DEE44), an autosomal recessive disorder characterized by early-onset encephalopathy, movement abnormalities, global developmental delay, intellectual disability, and seizures. DEE44 is caused by at least twelve different missense variants described as loss of function (LoF), but the relationships between genotypes and molecular or clinical phenotypes remains to be established.

Bacterial mimicry of eukaryotic HECT ubiquitin ligation.

HECT E3 ubiquitin (Ub) ligases direct their modified substrates toward a range of cellular fates dictated by the specific form of monomeric or polymeric Ub (polyUb) signal that is attached. How polyUb specificity is achieved has been a longstanding mystery, despite extensive study ranging from yeast to human. Two outlying examples of bacterial "HECT-like" (bHECT) E3 ligases have been reported in the human pathogens Enterohemorrhagic and Typhimurium, but what parallels can be drawn to eukaryotic HECT (eHECT) mechanism and specificity had not been explored.

Interplay between septins and ubiquitin-mediated xenophagy during entrapment.

Septins are cytoskeletal proteins implicated in numerous cellular processes including cytokinesis and morphogenesis. In the case of infection by , septins assemble into cage-like structures that entrap cytosolic bacteria targeted by autophagy. The interplay between septin cage entrapment and bacterial autophagy is poorly understood.

Septins and K63 ubiquitin chains are present in separate bacterial microdomains during autophagy of entrapped Shigella.

During host cell invasion, Shigella escapes to the cytosol and polymerizes actin for cell-to-cell spread. To restrict cell-to-cell spread, host cells employ cell-autonomous immune responses including antibacterial autophagy and septin cage entrapment. How septins interact with the autophagy process to target Shigella for destruction is poorly understood.

The acetylase activity of Cdu1 regulates bacterial exit from infected cells by protecting effectors from degradation.

Many cellular processes are regulated by ubiquitin-mediated proteasomal degradation. Pathogens can regulate eukaryotic proteolysis through the delivery of proteins with de-ubiquitinating (DUB) activities. The obligate intracellular pathogen secretes Cdu1 (ChlaDUB1), a dual deubiquitinase and Lys-acetyltransferase, that promotes Golgi remodeling and survival of infected host cells presumably by regulating the ubiquitination of host and bacterial proteins.

Bacterial usurpation of the OTU deubiquitinase fold.

The extensive cellular signalling events controlled by posttranslational ubiquitination are tightly regulated through the action of specialized proteases termed deubiquitinases. Among them, the OTU family of deubiquitinases can play very specialized roles in the regulation of discrete subtypes of ubiquitin signals that control specific cellular functions. To exert control over host cellular functions, some pathogenic bacteria have usurped the OTU deubiquitinase fold as a secreted virulence factor that interferes with ubiquitination inside infected cells.

Observing Real-Time Ubiquitination in High Throughput with Fluorescence Polarization.

Reconstitution of ubiquitin conjugation and deconjugation in vitro provides access to valuable information on enzyme kinetics, specificity, and structure-function relationships. Classically, these biochemical assays culminate in separation by SDS-PAGE and analysis by immunoblotting, an approach that requires additional time, can be difficult to quantify, and provides granular snapshots of the reaction progression. To address these limitations, we have implemented a fluorescence polarization-based assay that tracks ubiquitin conjugation and deconjugation in real time based upon changes in molecular weight.

Ester-linked ubiquitination by HOIL-1 controls immune signalling by shaping the linear ubiquitin landscape.

Ester-linked ubiquitination of serine or threonine residues - or even lipids - has emerged as a new regulatory earmark in cell signalling. Petrova et al. (2021) now reveal that ubiquitin esterification by the atypical ubiquitin ligase HOIL-1, a component of the LUBAC complex, is critical for proper formation of linear ubiquitin chains and control of immune signalling in T cells and macrophages.

Bacterial lipids earmarked with ubiquitin for pathogen clearance.

As a component of cell-autonomous immunity, cytosolic bacterial invaders are earmarked with the protein modifier ubiquitin for targeted xenophagic destruction. Otten et al. (2021) reveal that unique bacterial lipids are unconventional sites for ubiquitination and initiate downstream pathogen clearance.

Identification and characterization of diverse OTU deubiquitinases in bacteria.

Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The ovarian tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans.

The Tumour Suppressor TMEM127 Is a Nedd4-Family E3 Ligase Adaptor Required by Salmonella SteD to Ubiquitinate and Degrade MHC Class II Molecules.

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4 T cell activation.