Notes

Concordant serious myeloblastic leukemia in the exact same twin babies treated with allogeneic hair transplant coming from a more youthful HLA-identical brother or sister carrying out a one apheresis treatment.
One is a dual His6 and FLAG epitope-tagged version and the other is a fluorescent version where ALOD4 is fused to Neon, a monomeric fluorescent protein. These new forms of ALOD4 together with previously described OlyA provide an expanded collection of tools to sense, visualize, and modulate levels of accessible and SM-sequestered cholesterol on PMs and study the role of these cholesterol pools in diverse membrane signaling events.Very few proteins are reported to bind specific lipids. Because of the high selectivity and strong binding to specific lipids, lipid-targeting pore forming toxins (PFTs) have been employed to study the distribution of lipids in cell- and model-membranes. Non-toxic and monomeric PFT-derivatives are especially useful to study living cells. In this chapter we highlight sphingomyelin (SM)-binding PFT, lysenin (Lys), its derivatives, and newly identified SM/cholesterol binding protein, nakanori. We describe the preparation of non-toxic mutant of Lys (NT-Lys) and its application in optical and super resolution microscopy. We also discuss the observation of nanometer scale lipid domains labeled with nakanori and maltose-binding protein (MBP)-Lys in electron microscopy.Pore-forming proteins are found in prokaryotes, vertebrates, and invertebrates, and when involved in pathogenic processes they are classified as pore-forming toxins (PFTs). The use of gene engineering methods in combination with the information provided by the high-resolution crystal structures of the PFTs have allowed investigators to gain a deep understanding of their pore-forming mechanisms. In this chapter, we discuss how protein engineering has helped us and others to reveal the molecular mechanisms of pore formation by prokaryotic PFTs with an emphasis on our experiences with the cholesterol-dependent cytolysins (CDCs).Pore forming toxins (PFTs) are virulent proteins released by several species, including many strains of bacteria, to attack and kill host cells. In this article, we focus on the utility of molecular dynamics (MD) simulations and the molecular insights gleaned from these techniques on the pore forming pathways of PFTs. In addition to all-atom simulations which are widely used, coarse-grained MARTINI models and structure-based models have also been used to study PFTs. Here, the emphasis is on methods and techniques involved while setting up, monitoring, and evaluating properties from MD simulations of PFTs in a membrane environment. We draw from several case studies to illustrate how MD simulations have provided molecular insights into protein-protein and protein-lipid interactions, lipid dynamics, conformational transitions and structures of both the oligomeric intermediates and assembled pore structures.Single-channel recording from pore-forming toxins (PFTs) provides a clear and direct molecular readout of toxin action. However to complete any mechanistic understanding of PFT behavior, this functional kinetic readout must be linked to the underlying changes in toxin structure, binding, conformation, or stoichiometry. Here we review how single-molecule imaging methods might be used to further our understanding of PFTs, and provide detailed practical guidance on the use of droplet interface bilayers as a method capable of examining both single-molecule fluorescence and single-channel electrical signals from PFTs.PFPs (Pore-forming proteins) perforate cellular membranes to create an aqueous pore and allow the passage of ions and polar molecules. The molecular mechanisms for many of these PFPs have been elucidated by combining high resolution structural information of these proteins with biochemical and biophysical approaches. However, some PFPs do not adopt stable conformations and are difficult to study in vitro. An example of these proteins are the bacterial Type 3 Secretion (T3S) translocators. The translocators are secreted by the bacterium and insert into the target cell membrane to form a translocon pore providing a portal for the passage of T3S toxins into eukaryotic cells. Given the important role that the T3S systems play in pathogenesis, methods to study these translocon pores in cellular membranes are needed. Using a combination of protein modifications and methods to selectively permeate and solubilized eukaryotic membranes, we have established an experimental procedure to analyze the topology of the Pseudomonas aeruginosa T3S translocon using P. aeruginosa strain variants and HeLa cell lines.Mitochondria are important not only to healthy but also dying cells. In particular, apoptotic cell death initiates when the mitochondrial outer membrane is permeabilized by Bax, a protein of the Bcl-2 family. Bax shares a structural fold with some α-helical bacterial pore-forming toxins before these proteins actively engage membranes. Despite decades of intensive research, the structures of the pores formed by these proteins are mostly unknown, mainly because the pores are assembled by different numbers of the proteins whose conformation and interaction are highly dynamic. Site-specific crosslinking of the pore-forming proteins in cellular membranes where the pores are assembled is a powerful approach to assess the biological pore structure, dynamics and function. In this chapter, we describe a cysteine-based site-specific crosslinking protocol for the Bax protein in the mitochondrial membrane. We discuss the expected results and the resulting structural-functional models for the pore-forming Bax oligomer, in comparison with other crosslinking approaches that have been used to study other mitochondrial protein complexes. At the end, we highlight the advantages of the crosslinking approaches as well as the limitations and alternative approaches.Diphtheria toxin is among many bacterial toxins that utilize the endosomal pathway of cellular entry, which is ensured by the bridging of the endosomal membrane by the toxin's translocation (T) domain. Endosomal acidification triggers a series of conformational changes of the T-domain, that take place first in aqueous and subsequently in membranous milieu. Entinostat These rearrangements ultimately result in establishing membrane-inserted conformation(s) and translocation of the catalytic moiety of the toxin into the cytoplasm. We discuss here the strategy for combining site-selective labeling with various spectroscopic methods to characterize structural and thermodynamic aspects of protonation-dependent conformational switching and membrane insertion of the diphtheria toxin T-domain. Among the discussed methods are FRET, FCS and depth-dependent fluorescence quenching with lipid-attached bromine atoms and spin probes. The membrane-insertion pathway of the T-domain contains multiple intermediates and is governed by staggered pH-dependent transitions involving protonation of histidines and acidic residues.
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