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. We expanded the bHECT family with examples in human and plant pathogens. Three bHECT structures in primed, Ub-loaded states resolved key details of the entire Ub ligation process. One structure provided a rare glimpse into the act of ligating polyUb, yielding a means to rewire polyUb specificity of both bHECT and eHECT ligases. Studying this evolutionarily distinct bHECT family has revealed insight into the function of key bacterial virulence factors as well as fundamental principles underlying HECT-type Ub ligation.

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. Despite lacking a conventional ubiquitin system of their own, many bacterial ubiquitin regulators loosely follow the structural and mechanistic rules established by eukaryotic ubiquitin machinery. Others completely break these rules and have evolved novel structural folds, exhibit distinct mechanisms of regulation, or catalyze foreign ubiquitin modifications. Studying these interactions can not only reveal important aspects of bacterial pathogenesis but also shed light on unexplored areas of ubiquitin signaling and regulation. In this review, we discuss the methods by which bacteria manipulate host ubiquitin and highlight aspects that follow or break the rules of ubiquitination.

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 Escherichia coli and Salmonella Typhimurium, but what parallels can be drawn to eukaryotic HECT (eHECT) mechanism and specificity had not been explored. Here, we expanded the bHECT family and identified catalytically active, bona fide examples in both human and plant pathogens. By determining structures for three bHECT complexes in their primed, Ub-loaded states, we resolved key details of the full bHECT Ub ligation mechanism. One structure provided the first glimpse of a HECT E3 ligase in the act of ligating polyUb, yielding a means to rewire the polyUb specificity of both bHECT and eHECT ligases. Through studying this evolutionarily distinct bHECT family, we have not only gained insight into the function of key bacterial virulence factors but also revealed fundamental principles underlying HECT-type Ub ligation.

Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA.

The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential "adaptive" ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA's deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction.

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. We find this approach, which we have termed "UbiReal," to greatly facilitate biochemical studies such as mutational analyses, specificity determination, and inhibitor characterization.

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. Viral pathogens have also extensively co-opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin-bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and recognize a ubiquitin substrate. With these new examples, we perform the first cross-kingdom structural analysis of the OTU fold that highlights commonalities among distantly related OTU deubiquitinases.

A High-Throughput Assay for Monitoring Ubiquitination in Real Time.

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.

Biogas Biocatalysis: Methanotrophic Bacterial Cultivation, Metabolite Profiling, and Bioconversion to Lactic Acid.

Anaerobic digestion (AD) of waste substrates, and renewable biomass and crop residues offers a means to generate energy-rich biogas. However, at present, AD-derived biogas is primarily flared or used for combined heat and power (CHP), in part due to inefficient gas-to-liquid conversion technologies. Methanotrophic bacteria are capable of utilizing methane as a sole carbon and energy source, offering promising potential for biological gas-to-liquid conversion of AD-derived biogas. Here, we report cultivation of three phylogenetically diverse methanotrophic bacteria on biogas streams derived from AD of a series of energy crop residues. Strains maintained comparable central metabolic activity and displayed minimal growth inhibition when cultivated under batch configuration on AD biogas streams relative to pure methane, although metabolite analysis suggested biogas streams increase cellular oxidative stress. In contrast to batch cultivation, growth arrest was observed under continuous cultivation configuration, concurrent with increased biosynthesis and excretion of lactate. We examined the potential for enhanced lactate production via the employ of a pyruvate dehydrogenase mutant strain, ultimately achieving 0.027 g lactate/g DCW/h, the highest reported lactate specific productivity from biogas to date.