Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis.

Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact EV-based analysis and biomarker profiling, we investigated the presence and formation of EV-LPP complexes in biological samples by using label-free atomic force microscopy, cryo-electron tomography and synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high sensitivity single particle flow cytometry. Furthermore, we evaluated the impact on flow cytometric analysis in the presence of LPPs using in vitro spike-in experiments of purified tumour cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrate that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations, can influence quantitative and qualitative EV analysis. Lastly, we demonstrate that the colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis and EV biomarker profiling.

The crystal structure of the EspB-EspK virulence factor-chaperone complex suggests an additional type VII secretion mechanism in Mycobacterium tuberculosis.

Pathogenic species from the Mycobacterium genus are responsible for a number of adverse health conditions in humans and animals that threaten health security and the economy worldwide. Mycobacteria have up to five specialized secretion systems (ESX-1 to ESX-5) that transport virulence factors across their complex cell envelope to facilitate manipulation of their environment. In pathogenic species, these virulence factors influence the immune system's response and are responsible for membrane disruption and contributing to cell death. While structural details of these secretion systems have been recently described, gaps still remain in the structural understanding of the secretion mechanisms of most substrates. Here, we describe the crystal structure of Mycobacterium tuberculosis ESX-1 secretion-associated substrate EspB bound to its chaperone EspK. We found that EspB interacts with the C-terminal domain of EspK through its helical tip. Furthermore, cryogenic electron microscopy, size exclusion chromatography analysis, and small-angle X-ray scattering experiments show that EspK keeps EspB in its secretion-competent monomeric form and prevents its oligomerization. The structure presented in this study suggests an additional secretion mechanism in ESX-1, analogous to the chaperoning of proline-glutamate (PE)-proline-proline-glutamate (PPE) proteins by EspG, where EspK facilitates the secretion of EspB in Mycobacterium species.

Priming mycobacterial ESX-secreted protein B to form a channel-like structure.

ESX-1 is a major virulence factor of Mycobacterium tuberculosis, a secretion machinery directly involved in the survival of the microorganism from the immune system defence. It disrupts the phagosome membrane of the host cell through a contact-dependent mechanism. Recently, the structure of the inner-membrane core complex of the homologous ESX-3 and ESX-5 was resolved; however, the elements involved in the secretion through the outer membrane or those acting on the host cell membrane are unknown. Protein substrates might form this missing element. Here, we describe the oligomerisation process of the ESX-1 substrate EspB, which occurs upon cleavage of its C-terminal region and is favoured by an acidic environment. Cryo-electron microscopy data shows that quaternary structure of EspB is conserved across slow growing species, but not in the fast growing M. smegmatis. EspB assembles into a channel with dimensions and characteristics suitable for the transit of ESX-1 substrates, as shown by the presence of another EspB trapped within. Our results provide insight into the structure and assembly of EspB, and suggests a possible function as a structural element of ESX-1.

Mycobacterium tuberculosis ferritin: a suitable workhorse protein for cryo-EM development.

The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol is presented that provides an apoferritin, bacterioferritin B (BfrB), from Mycobacterium tuberculosis with high yield and purity. A 2.12 Šresolution cryo-EM structure of BfrB is reported, showing the typical cage-like oligomer constituting of 24 monomers related by 432 symmetry. However, it also contains a unique C-terminal extension (164-181), which loops into the cage region of the shell and provides extra stability to the protein. Part of this region was ambiguous in previous crystal structures but could be built within the cryo-EM map. These findings and this protocol could serve the growing cryo-EM community in characterizing and pushing the limits of their electron microscopes and workflows.

Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification.

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

Exploring the role of elongation Factor-Like 1 (EFL1) in Shwachman-Diamond syndrome through molecular dynamics.

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder whose patients present mutations in two ribosome assembly proteins, the Shwachman-Bodian-Diamond Syndrome protein (SBDS) and the Elongation Factor-Like 1 (EFL1). Due to the lack of knowledge of the molecular mechanisms responsible for SDS pathogenesis, current therapy is nonspecific and focuses only at alleviating the symptoms. Building on the recent observation that EFL1 single-point mutations clinically manifest as SDS-like phenotype, we carried out comparative Molecular Dynamics (MD) simulations on three mutants, T127A, M882K and R1095Q and wild type EFL1. As supported by small angle X-ray scattering experiments, the obtained data improve the static EFL1 model resulting from the Cryo-electron microscopy and clearly show that all the mutants experience a peculiar rotation, around the hinge region, of domain IV with respect to domains I and II leading to a different conformation respect to that of wild type protein. This study supports the notion that EFL1 function is governed by an allosteric mechanism involving the concerted action of GTPase domain (domain I) and the domain IV and can help point towards new approaches to SDS treatment.Communicated by Ramaswamy H. Sarma.

Interaction of the GTPase Elongation Factor Like-1 with the Shwachman-Diamond Syndrome Protein and Its Missense Mutations.

The Shwachman-Diamond Syndrome (SDS) is a disorder arising from mutations in the genes encoding for the Shwachman-Bodian-Diamond Syndrome (SBDS) protein and the GTPase known as Elongation Factor Like-1 (EFL1). Together, these proteins remove the anti-association factor eIF6 from the surface of the pre-60S ribosomal subunit to promote the formation of mature ribosomes. SBDS missense mutations can either destabilize the protein fold or affect surface epitopes. The molecular alterations resulting from the latter remain largely unknown, although some evidence suggest that binding to EFL1 may be affected. We further explored the effect of these SBDS mutations on the interaction with EFL1, and showed that all tested mutations disrupted the binding to EFL1. Binding was either severely weakened or almost abolished, depending on the assessed mutation. In higher eukaryotes, SBDS is essential for development, and lack of the protein results in early lethality. The existence of patients whose only source of SBDS consists of that with surface missense mutations highlights the importance of the interaction with EFL1 for their function. Additionally, we studied the interaction mechanism of the proteins in solution and demonstrated that binding consists of two independent and cooperative events, with domains 2⁻3 of SBDS directing the initial interaction with EFL1, followed by docking of domain 1. In solution, both proteins exhibited large flexibility and consisted of an ensemble of conformations, as demonstrated by Small Angle X-ray Scattering (SAXS) experiments.

Mutations in EFL1, an SBDS partner, are associated with infantile pancytopenia, exocrine pancreatic insufficiency and skeletal anomalies in aShwachman-Diamond like syndrome.

BACKGROUND: For the final step of the maturation of the ribosome, the nascent 40S and 60S subunits are exported from the nucleus to the cell cytoplasm. To prevent premature association of these ribosomal subunits, eukaryotic initiation factor 6 (eIF6) binds the 60S subunit within the nucleus. Its release in the cytoplasm requires the interaction of EFL1 and SDBS proteins. In Shwachman-Diamond syndrome (SDS), a defective SDBS protein prevents eIF6 eviction, inhibiting its recycle to the nucleus and subsequent formation of the active 80S ribosome. OBJECTIVE: This study aims to identify the molecular basis of an SDS-like disease, manifested by pancytopenia, exocrine pancreatic insufficiency and skeletal abnormalities in six patients from three unrelated families. METHODS: Whole exome analysis was used for mutation identification. Fluorescence microscopy studies assessed the localisation of Tif6-GFP, the yeast eIF6 homologue, in yeast WT and mutant cells. Human and yeast EFL1 proteins, WT and mutants, were expressed in Saccharomyces cerevisiae BCY123 strain, and circular dichroism and small-angle X-ray scattering were used to assess the folding and flexibility of these proteins. Green malachite colorimetric assay was performed to determine the GTPase activity of WT and Efl1 mutants. RESULTS: Four patients were homozygous for p.R1095Q variant and two patients were homozygous for p.M882K variant in EFL1. Residue R1095 and M882 are conserved across species. Neither the GTPase activity of the mutant proteins nor its activation by the SDBD protein or the 60S ribosomal subunit were affected. Complementation of efl1Δ yeast cells with the EFL1 mutants rescued the slow growth phenotype. Nonetheless, Tif6-GFP was relocalised to the cytoplasm in mutant yeast cells in contrast to its nuclear localisation in WT cells. CONCLUSIONS: Mutations in EFL1 clinically manifest as SDS-like phenotype. Similar to the molecular pathology of SDS, mutant EFL1 proteins do not promote the release of cytoplasmic Tif6 from the 60S subunit, likely preventing the formation of mature ribosomes.

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions.

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4',5'-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein.

Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1.

Guanine nucleotide exchange in the ribosomal GTPase EFL1 is modulated by the protein mutated in the Shwachman-Diamond syndrome.

Ribosome biogenesis in eukaryotes is a complex process that requires the participation of several accessory proteins that are not part of the mature particle. Efl1 is a yeast GTPase required for the cytoplasmic maturation of the 60S ribosomal subunit. Together with Sdo1, the yeast ortholog of the protein mutated in the Shwachman-Diamond Syndrome (SBDS), Efl1 releases the anti-association factor Tif6 from the surface of the 60S subunit allowing the assembly of mature ribosomes. We characterized the structural content and folding stability of the Saccharomyces cerevisiae and human EFL1 GTPases, as well as their enzymatic properties alone and in the presence of Sdo1 and SBDS, respectively. The human and S. cerevisiae EFL1 GTPases are composed of a mixture of α-helices and ß-sheets. Despite being orthologs, the yeast protein elicited a non-two state thermal unfolding behavior while the human EFL1 was highly resistant to thermal denaturation. Steady-state kinetic analyses indicated slow GTP hydrolysis for both EFL1 GTPases, with kcat values of 0.4 and 0.3min(-1) and Km for GTP of 110 and 180µM respectively. In the presence of the effector proteins, their kcat values remained unaltered while the Km decreased twofold suggesting that Sdo1 and SBDS act as nucleotide exchange factors.