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Potent macromolecule-sized poration of lipid bilayers by the macrolittins, a synthetically evolved family of pore-forming peptides.

J Am Chem Soc. 2018 Apr 25;:

Authors: Li S, Kim SY, Pittman AE, King GM, Wimley WC, Hristova K

Abstract
Potent macromolecule-sized poration of lipid bilayers by the macrolittins, a synthetically evolved family of pore-forming peptides. Sijia Li1, Sarah Y. Kim1, Anna E. Pittman3, Gavin M. King3,4, William C. Wimley2* and Kalina Hristova1* 1Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218 2Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA 70112 3Physics and Astronomy, University of Missouri, Columbia, MO, 65201 4Biochemistry, University of Missouri, Columbia, MO, 65201 *Address correspondence to wwimley@tulane.edu and kh@jhu.edu   Abstract Pore-forming peptides with novel functions have potential utility in many biotechnological applications. However, the sequence-structure-function relationships of pore forming peptides are not understood well enough to empower rational design. Therefore, in this work we used synthetic molecular evolution to identify a novel family of peptides that are highly potent and cause macromolecular poration in synthetic lipid vesicles at low peptide concentration and at neutral pH. These unique 26-residue peptides, which we call macrolittins, release macromolecules from lipid bilayer vesicles made from zwitterionic PC lipids at peptide to lipid ratios as low as 1:1000, a property that is almost unprecedented among known membrane permeabilizing peptides. The macrolittins exist as membrane-spanning -helices. They cause dramatic bilayer thinning and form large pores in planar supported bilayers. The high potency of these peptides is likely due to their ability to stabilize bilayer edges by a process that requires specific electrostatic interactions between peptides.

PMID: 29694775 [PubMed - as supplied by publisher]

Phosphotyrosine phosphatase R3 receptors: Origin, evolution and structural diversification.

PLoS One. 2017;12(3):e0172887

Authors: Chicote JU, DeSalle R, García-España A

Abstract
Subtype R3 phosphotyrosine phosphatase receptors (R3 RPTPs) are single-spanning membrane proteins characterized by a unique modular composition of extracellular fibronectin repeats and a single cytoplasmatic protein tyrosine phosphatase (PTP) domain. Vertebrate R3 RPTPs consist of five members: PTPRB, PTPRJ, PTPRH and PTPRO, which dephosphorylate tyrosine residues, and PTPRQ, which dephosphorylates phophoinositides. R3 RPTPs are considered novel therapeutic targets in several pathologies such as ear diseases, nephrotic syndromes and cancer. R3 RPTP vertebrate receptors, as well as their known invertebrate counterparts from animal models: PTP52F, PTP10D and PTP4e from the fruitfly Drosophila melanogaster and F44G4.8/DEP-1 from the nematode Caenorhabditis elegans, participate in the regulation of cellular activities including cell growth and differentiation. Despite sharing structural and functional properties, the evolutionary relationships between vertebrate and invertebrate R3 RPTPs are not fully understood. Here we gathered R3 RPTPs from organisms covering a broad evolutionary distance, annotated their structure and analyzed their phylogenetic relationships. We show that R3 RPTPs (i) have probably originated in the common ancestor of animals (metazoans), (ii) are variants of a single ancestral gene in protostomes (arthropods, annelids and nematodes); (iii) a likely duplication of this ancestral gene in invertebrate deuterostomes (echinodermes, hemichordates and tunicates) generated the precursors of PTPRQ and PTPRB genes, and (iv) R3 RPTP groups are monophyletic in vertebrates and have specific conserved structural characteristics. These findings could have implications for the interpretation of past studies and provide a framework for future studies and functional analysis of this important family of proteins.

PMID: 28257417 [PubMed - in process]

Abstract

Nature Chemistry 8, 997 (2016). doi:10.1038/nchem.2629

Authors: Alexander N. Zelikin, Carsten Ehrhardt & Anne Marie Healy

Biological drugs can offer high potency and selectivity; however, this class of therapeutics often shows poor stability upon oral administration and during subsequent circulation. This Review highlights the materials and methods used to deliver biological drugs, and discusses how these approaches can improve their pharmacokinetics.

The Chlamydia pneumoniae Adhesin Pmp21 forms Oligomers with Adhesive Properties.

J Biol Chem. 2016 Aug 22;

Authors: Luczak SE, Smits SH, Decker C, Nagel-Steger L, Schmitt L, Hegemann JH

Abstract
Chlamydiae sp. are obligate intracellular pathogens that cause a variety of diseases in humans. Adhesion of Chlamydiae to the eukaryotic host cell is a pivotal step in pathogenesis. The adhesin family of polymorphic membrane proteins (Pmp) in Chlamydia pneumoniae consists of 21 members. Pmp21 binds to the epidermal growth factor receptor (EGFR). Pmps contain large numbers of FxxN and GGA(I, L, V) motifs. At least two of these motifs are crucial for adhesion by certain Pmp21 fragments. Here we describe how the two FxxN motifs in Pmp21-D (D-Wt), a domain of Pmp21, influence its self-interaction, folding and adhesive capacities. Refolded D-Wt molecules form oligomers with high sedimentation values (8 to 85 S). These oligomers take the form of elongated protofibrils which exhibit Thioflavin-T fluorescence, like the amyloid protein fragment β42. A mutant version of Pmp21-D (D-Mt), with FxxN motifs replaced by SxxV, shows a markedly reduced capacity to form oligomers. Secondary-structure assays revealed that monomers of both variants exist predominantly as random coils, whereas the oligomers form predominantly β-sheets. Adhesion studies revealed that oligomers of D-Wt (D-Wt-O) mediated significantly enhanced binding to human epithelial cells relative to D-Mt-O and monomeric protein species. Moreover, D-Wt-O bound EGFR more efficiently than D-Wt monomers. Importantly, pre-treatment of human cells with D-Wt-O reduces infectivity upon subsequent challenge with C. pneumoniae more effectively than all other protein species. Hence, the FxxN motif in D-Wt induces the formation of β-sheet-rich oligomeric protofibrils which are important for adhesion to, and subsequent infection of human cells.

PMID: 27551038 [PubMed - as supplied by publisher]

Lactase nonpersistence is directed by DNA-variation-dependent epigenetic aging.

Nat Struct Mol Biol. 2016 May 9;

Authors: Labrie V, Buske OJ, Oh E, Jeremian R, Ptak C, Gasiūnas G, Maleckas A, Petereit R, Žvirbliene A, Adamonis K, Kriukienė E, Koncevičius K, Gordevičius J, Nair A, Zhang A, Ebrahimi S, Oh G, Šikšnys V, Kupčinskas L, Brudno M, Petronis A

Abstract
The inability to digest lactose, due to lactase nonpersistence, is a common trait in adult mammals, except in certain human populations that exhibit lactase persistence. It is not known how the lactase gene is dramatically downregulated with age in most individuals but remains active in some individuals. We performed a comprehensive epigenetic study of human and mouse small intestines, by using chromosome-wide DNA-modification profiling and targeted bisulfite sequencing. Epigenetically controlled regulatory elements accounted for the differences in lactase mRNA levels among individuals, intestinal cell types and species. We confirmed the importance of these regulatory elements in modulating lactase mRNA levels by using CRISPR-Cas9-induced deletions. Genetic factors contribute to epigenetic changes occurring with age at the regulatory elements, because lactase-persistence and lactase-nonpersistence DNA haplotypes demonstrated markedly different epigenetic aging. Thus, genetic factors enable a gradual accumulation of epigenetic changes with age, thereby influencing phenotypic outcome.

PMID: 27159559 [PubMed - as supplied by publisher]

Abstract

Nature Chemical Biology. doi:10.1038/nchembio.2061

Authors: Joshua A Baccile, Joseph E Spraker, Henry H Le, Eileen Brandenburger, Christian Gomez, Jin Woo Bok, Juliane Macheleidt, Axel A Brakhage, Dirk Hoffmeister, Nancy P Keller & Frank C Schroeder

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Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface.

J Biol Chem. 2015 Oct 16;290(42):25728-44

Authors: Jastrzebska B, Chen Y, Orban T, Jin H, Hofmann L, Palczewski K

Abstract
Although homo- and heterodimerizations of G protein-coupled receptors (GPCRs) are well documented, GPCR monomers are able to assemble in different ways, thus causing variations in the interactive interface between receptor monomers among different GPCRs. Moreover, the functional consequences of this phenomenon, which remain to be clarified, could be specific for different GPCRs. Synthetic peptides derived from transmembrane (TM) domains can interact with a full-length GPCR, blocking dimer formation and affecting its function. Here we used peptides corresponding to TM helices of bovine rhodopsin (Rho) to investigate the Rho dimer interface and functional consequences of its disruption. Incubation of Rho with TM1, TM2, TM4, and TM5 peptides in rod outer segment (ROS) membranes shifted the resulting detergent-solubilized protein migration through a gel filtration column toward smaller molecular masses with a reduced propensity for dimer formation in a cross-linking reaction. Binding of these TM peptides to Rho was characterized by both mass spectrometry and a label-free assay from which dissociation constants were calculated. A BRET (bioluminescence resonance energy transfer) assay revealed that the physical interaction between Rho molecules expressed in membranes of living cells was blocked by the same four TM peptides identified in our in vitro experiments. Although disruption of the Rho dimer/oligomer had no effect on the rates of G protein activation, binding of Gt to the activated receptor stabilized the dimer. However, TM peptide-induced disruption of dimer/oligomer decreased receptor stability, suggesting that Rho supramolecular organization could be essential for ROS stabilization and receptor trafficking.

PMID: 26330551 [PubMed - indexed for MEDLINE]

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Mechanism of FGF receptor dimerization and activation.

Nat Commun. 2016;7:10262

Authors: Sarabipour S, Hristova K

Abstract
Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

PMID: 26725515 [PubMed - indexed for MEDLINE]

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A lysine-rich motif in the phosphatidylserine receptor PSR-1 mediates recognition and removal of apoptotic cells.

Nat Commun. 2015;6:5717

Authors: Yang H, Chen YZ, Zhang Y, Wang X, Zhao X, Godfroy JI, Liang Q, Zhang M, Zhang T, Yuan Q, Ann Royal M, Driscoll M, Xia NS, Yin H, Xue D

Abstract
The conserved phosphatidylserine receptor (PSR) was first identified as a receptor for phosphatidylserine, an 'eat-me' signal exposed by apoptotic cells. However, several studies suggest that PSR may also act as an arginine demethylase, a lysyl hydroxylase, or an RNA-binding protein through its N-terminal JmjC domain. How PSR might execute drastically different biochemical activities, and whether they are physiologically significant, remain unclear. Here we report that a lysine-rich motif in the extracellular domain of PSR-1, the Caenorhabditis elegans PSR, mediates specific phosphatidylserine binding in vitro and clearance of apoptotic cells in vivo. This motif also mediates phosphatidylserine-induced oligomerization of PSR-1, suggesting a mechanism by which PSR-1 activates phagocytosis. Mutations in the phosphatidylserine-binding motif, but not in its Fe(II) binding site critical for the JmjC activity, abolish PSR-1 phagocytic function. Moreover, PSR-1 enriches and clusters around apoptotic cells during apoptosis. These results establish that PSR-1 is a conserved, phosphatidylserine-recognizing phagocyte receptor.

PMID: 25564762 [PubMed - indexed for MEDLINE]

Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

J Am Chem Soc. 2015 Oct 30;

Authors: Koldsø H, Sansom MS

Abstract
The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high resolution microscopy can reveal the organisation and dynamics of proteins and lipids within living cells at resolutions < 200 nm. Parallel advances in molecular simulations provide near-atomic resolution models of the dynamics of the organisation of membranes of in vivo like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the length and complexity gap between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20 to 100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

PMID: 26517394 [PubMed - as supplied by publisher]

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Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

Nucleic Acids Res. 2015 Jul 27;43(13):6579-86

Authors: Kim S, Zbaida D, Elbaum M, Leh H, Nogues C, Buckle M

Abstract
VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1.

PMID: 26044711 [PubMed - indexed for MEDLINE]