Mechanism of effector capture and delivery by the type IV secretion system from Legionella pneumophila

Legionella pneumophila is a bacterial pathogen that utilises a Type IV secretion (T4S) system to inject effector proteins into human macrophages. Essential to the recruitment and delivery of effectors to the T4S machinery is the membrane-embedded T4 coupling complex (T4CC). Here, we purify an intact T4CC from the Legionella membrane. It contains the DotL ATPase, the DotM and DotN proteins, the chaperone module IcmSW, and two previously uncharacterised proteins, DotY and DotZ. The atomic resolution structure reveals a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module protrudes. Six of these hetero-pentameric complexes may assemble into a 1.6-MDa hexameric nanomachine, forming an inner membrane channel for effectors to pass through. Analysis of multiple cryo EM maps, further modelling and mutagenesis provide working models for the mechanism for binding and delivery of two essential classes of Legionella effectors, depending on IcmSW or DotM, respectively

The proteins DotY and DotZ modulate the dynamics and localization of the type IVB coupling complex of Legionella pneumophila

Legionella pneumophila is an opportunistic pathogen infecting alveolar macrophages and protozoa species. Legionella utilizes a Type IV Secretion System (T4SS) to translocate over 300 effector proteins into its host cell. In a recent study, we isolated and solved the cryo-EM structure of the Type IV Coupling Complex (T4CC), a large cytoplasmic determinant associated with the inner membrane that recruits effector proteins for delivery to the T4SS for translocation. The T4CC is composed of a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module flexibly protrudes. The DotY and DotZ proteins were newly reported members of this complex and their role remained elusive. In this study, we observed the effect of deleting DotY and DotZ on T4CC stability and localization. Furthermore, we found these two proteins are co-dependent, whereby the deletion of DotY resulted in DotZ absence from the coupling complex, and vice versa. Additional cryo-EM data analysis revealed the dynamic movement of the IcmSW module is modified by the DotY/Z proteins. We therefore determined the likely function of DotY and DotZ and revealed their importance on T4CC function

Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types in polyubiquitin co-exist and are independently regulated in cells. This ‘ubiquitin code’ determines the fate of the modified protein1. Deubiquitinating enzymes of the Ovarian Tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin2, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. We here reveal how the deubiquitinase Cezanne/OTUD7B specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with mono- and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable reconstruction of the enzymatic cycle in exquisite detail. An intricate mechanism of ubiquitin-assisted conformational changes activate the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the fascinating plasticity of deubiquitinases, and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site

Cryptococcal Neuroradiological Lesions Correlate with Severity during Cryptococcal Meningoencephalitis in HIV-Positive Patients in the HAART Era

Cryptococcal meningoencephalitis has an overall global mortality rate of 20% in AIDS patients despite antifungals. There is a need for additional means of precise assessment of disease severity. We thus studied the radiological brain images available from 62 HIV-positive patients with cryptococcocal meningoencephalitis to analyse the brain lesions associated with cryptococcosis in relationship with disease severity, and the respective diagnostic contribution of magnetic resonance (MR) versus computed tomography (CT)

Plasma and cellular fibronectin: distinct and independent functions during tissue repair

Fibronectin (FN) is a ubiquitous extracellular matrix (ECM) glycoprotein that plays vital roles during tissue repair. The plasma form of FN circulates in the blood, and upon tissue injury, is incorporated into fibrin clots to exert effects on platelet function and to mediate hemostasis. Cellular FN is then synthesized and assembled by cells as they migrate into the clot to reconstitute damaged tissue. The assembly of FN into a complex three-dimensional matrix during physiological repair plays a key role not only as a structural scaffold, but also as a regulator of cell function during this stage of tissue repair. FN fibrillogenesis is a complex, stepwise process that is strictly regulated by a multitude of factors. During fibrosis, there is excessive deposition of ECM, of which FN is one of the major components. Aberrant FN-matrix assembly is a major contributing factor to the switch from normal tissue repair to misregulated fibrosis. Understanding the mechanisms involved in FN assembly and how these interplay with cellular, fibrotic and immune responses may reveal targets for the future development of therapies to regulate aberrant tissue-repair processes

Neutronenstreuexperimente zur mikroskopischen Dynamik in ungeordneten Polymersystemen

Adhesive chaperone-usher pili are long, supramolecular protein fibers displayed on the surface of many bacterial pathogens. The type 1 and P pili of uropathogenic Escherichia coli (UPEC) play important roles during urinary tract colonization, mediating attachment to the bladder and kidney, respectively. The biomechanical properties of the helical pilus rods allow them to reversibly uncoil in response to flow-induced forces, allowing UPEC to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2-Å resolution cryo-EM structure of the type 1 pilus rod, which together with the previous P pilus rod structure rationalizes the remarkable “spring-like” properties of chaperone-usher pili. The cryo-EM structure of the type 1 pilus rod differs in its helical parameters from the structure determined previously by a hybrid approach. We provide evidence that these structural differences originate from different quaternary structures of pili assembled in vivo and in vitro

Monitoring RIPK1 phosphorylation in the TNFR1 signaling complex

Receptor-interacting protein kinase 1 (RIPK1) is a component of the TNFR1 signaling complex (also known as complex I or TNFR-SC), where its ubiquitylation by cIAP1/2 and LUBAC serves to initiate prosurvival and proinflammatory responses through activation of the MAPK and NF-κB pathways. IKKα/β-mediated phosphorylation of RIPK1 in complex I was shown to maintain RIPK1 in a prosurvival modus. Consequently, conditions affecting proper IKKα/β activation perturb IKKα/β-phosphorylation of RIPK1 and switch the TNF response toward RIPK1 kinase-dependent cell death. Methods to study the posttranslational modifications of RIPK1 in complex I are therefore of great value. Here, we describe a detailed protocol to isolate complex I-associated RIPK1 from cells and provide different tools to study the phosphorylation status of RIPK1 in TNFR1 complex I

Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest)

Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (multiple linkage types per polymer) chains poses significant problems for biochemical analysis. Here we describe UbiCRest, in which substrates (ubiquitinated proteins or polyubiquitin chains) are treated with a panel of linkage-specific deubiquitinating enzymes (DUBs) in parallel reactions, followed by gel-based analysis. UbiCRest can be used to show that a protein is ubiquitinated, to identify which linkage type(s) are present on polyubiquitinated proteins and to assess the architecture of heterotypic polyubiquitin chains. DUBs used in UbiCRest can be obtained commercially; however, we include details for generating a toolkit of purified DUBs and for profiling their linkage preferences in vitro. UbiCRest is a qualitative method that yields insights into ubiquitin chain linkage types and architecture within hours, and it can be performed on western blotting quantities of endogenously ubiquitinated proteins