Search

Septins are cytoskeletal proteins implicated in numerous cellular processes including cytokinesis and morphogenesis. In the case of infection by Shigella flexneri, 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. We used a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study septin cage entrapment of Shigella in its near-native state. Septin cages could be identified as X-ray dense structures, indicating they contain host cell proteins and lipids consistent with their autophagy links. Airyscan confocal microscopy of Shigella-septin cages showed that septins and lysine 63 (K63)-linked ubiquitin chains are present in separate bacterial microdomains, suggesting they are recruited separately. Finally, Cryo-SXT and live-cell imaging revealed an interaction between septins and microtubule-associated protein light chain 3B (LC3B)-positive membranes during autophagy of Shigella. Collectively our data present a new model for how septin-caged Shigella are targeted to autophagy.

Protein ubiquitination is a highly orchestrated process that controls diverse aspects of human biology. Dysregulation of this process can lead to various disease states including cancer, neurodegeneration, and autoimmunity. It is the correction of these dysregulated pathways, as well as the enticing ability to manipulate protein stability, that have instigated intense research into the therapeutic control of protein ubiquitination. A major bottleneck in the development and validation of small molecule modulators is the availability of a suitable high-throughput assay for enzyme activity. Herein, we present a new assay, which we term UbiReal, that uses fluorescence polarization to monitor all stages of Ub conjugation and deconjugation in real time. We use the assay to validate a chemical inhibitor of the E1 ubiquitin-activating enzyme, as well as to assess the activities and specificities of E2s, E3s, and deubiquitinases. The sensitivity and accessibility of this approach make it an excellent candidate for high-throughput screens of activity modulators, as well as a valuable tool for basic research into the mechanisms of ubiquitin regulation.

Background Although pathogenic Gram-negative bacteria lack their own ubiquitination machinery, they have evolved or acquired virulence effectors that can manipulate the host ubiquitination process through structural and/or functional mimicry of host machinery. Many such effectors have been identified in a wide variety of bacterial pathogens that share little sequence similarity amongst themselves or with eukaryotic ubiquitin E3 ligases. Methods To allow identification of novel bacterial E3 ubiquitin ligase effectors from protein sequences we have developed a machine learning approach, the SVM-based Identification and Evaluation of Virulence Effector Ubiquitin ligases (SIEVE-Ub). We extend the string kernel approach used previously to sequence classification by introducing reduced amino acid (RED) alphabet encoding for protein sequences. Results We found that 14mer peptides with amino acids represented as simply either hydrophobic or hydrophilic provided the best models for discrimination of E3 ligases from other effector proteins with a receiver-operator characteristic area under the curve (AUC) of 0.90. When considering a subset of E3 ubiquitin ligase effectors that do not fall into known sequence based families we found that the AUC was 0.82, demonstrating the effectiveness of our method at identifying novel functional family members. Feature selection was used to identify a parsimonious set of 10 RED peptides that provided good discrimination, and these peptides were found to be located in functionally important regions of the proteins involved in E2 and host target protein binding. Our general approach enables construction of models based on other effector functions. We used SIEVE-Ub to predict nine potential novel E3 ligases from a large set of bacterial genomes. SIEVE-Ub is available for download at https://doi.org/10.6084/m9.figshare.7766984.v1 or https://github.com/biodataganache/SIEVE-Ub for the most current version.

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 Chlamydia trachomatis 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. Here, we determined that Cdu1’s acetylase but not its DUB activity is important to protect Cdu1 from ubiquitin-mediated degradation. We further identified three C. trachomatis proteins on the pathogen-containing vacuole (InaC, IpaM, and CTL0480) that required Cdu1‘s acetylase activity for protection from degradation and determined that Cdu1 and these Cdu1-protected proteins are required for optimal egress of Chlamydia from host cells. These findings highlight a non-canonical mechanism of pathogen-mediated protection of virulence factors from degradation after their delivery into host cells and the coordinated regulation of secreted effector proteins.

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and endoplasmic reticulum stress. Variants in the UBA5 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. We developed a humanized UBA5 fly model and biochemical activity assays in order to describe in vivo and in vitro genotype–phenotype relationships across the UBA5 allelic series. In vivo, we observed a broad spectrum of phenotypes in viability, developmental timing, lifespan, locomotor activity, and bang sensitivity. A range of functional effects was also observed in vitro across comprehensive biochemical assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation. Importantly, there is a strong correlation between in vivo and in vitro phenotypes, establishing a classification of LoF variants into mild, intermediate, and severe allelic strengths. By systemically evaluating UBA5 variants across in vivo and in vitro platforms, this study provides a foundation for more basic and translational UBA5 research, as well as a basis for evaluating current and future individuals afflicted with this rare disease.