Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), ...consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues 90GLALIVAGV98); and (ii) a peptide comprised of the full TM4(85–105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88–100), and then “staple” this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-88VVGLXLIZXGVVV100-KKK-NH2 (X = 2-(4′-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn95 peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88–100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes.Helix-helix interactions commonly mediate a host of biological functions in membrane proteins.
A stapled peptide sequentially complementary to a helix-helix interaction motif disrupted a bacterial multidrug resistance efflux pump.
Stapling membrane-penetrating peptides represents a viable way to target protein-protein interactions within membranes while retaining metabolic stability.
Stapled peptides are prospective therapeutics for inhibiting membrane protein interactions involved in disease states.
Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point ...mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations – glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis.
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•CF-phenotypic loop mutants affect CFTR TM3/4 hairpin interactions with detergent.•Mutants at E217 and Q220 display wide variations in helicity and SDS-PAGE migration.•Mutant maturation levels of the corresponding full-length CFTRs were reduced vs. WT.•Point mutations evoke changes in protein-protein and/or protein-lipid interactions.
Insertion of a nascent membrane protein segment by the translocon channel into the bilayer is naturally promoted by high segmental hydrophobicity, but its selection as a transmembrane (TM) segment is ...complicated by the diverse environments (aqueous vs lipidic) the protein encounters and by the fact that most TM segments contain a substantial amount (∼30%) of polar residues, as required for protein structural stabilization and/or function. To examine the contributions of these factors systematically, we designed and synthesized a peptide library consisting of pairs of compositionally identical, but sequentially different, peptides with 19-residue core sequences varying (i) in Leu positioning (with five or seven Leu residues clustered into a contiguous “block” in the middle of the segment or “scrambled” throughout the sequence) and (ii) in Ser content (0–6 residues). The library was analyzed by a combination of biophysical and biological techniques, including HPLC retention times, circular dichroism measurements of helicity in micelle and phospholipid bilayer media, and relative blue shifts in Trp fluorescence maxima, as well as by the extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum. We found that local blocks of high hydrophobicity heighten the translocon’s propensity to insert moderately hydrophilic sequences, until a “threshold hydrophilicity” is surpassed whereby segments no longer insert even in the presence of Leu blocks. This study codifies the prerequisites of apolar/polar content and residue positioning that define nascent TM segments, illustrates the accuracy in their prediction, and highlights how a single disease-causing mutation can tip the balance toward anomalous translocation/insertion.
Large, hydrophobic residues (isoleucine, leucine, and valine) dominate sequences of transmembrane (TM) helices in membrane proteins (total ∼34%), but their relative roles in mediating the ...biologically relevant protein–lipid and protein–protein interactions have not been systematically evaluated. Here we have synthesized Leu‐containing Lys‐tagged hydrophobic peptides of identical composition, where sequences have been designed with their Leu residues either scrambled (sequence KKKLAASALAAAWLAALALSAAKKK); clustered (KKKAAASAALLLWLLAAAASAAKKK); or “lipopathic” (all Leu on one helical face) (KKKAAASLAALLWALLAAASAAKKK). These peptides were compared by several biophysical/biochemical techniques to the corresponding set of peptides where the Leu residues are replaced by the isosteric Ile residues. Circular dichroism spectra showed that all peptides were helical in POPC liposomes, as confirmed by blue shifts in Trp fluorescence spectra, notably with the Ile‐lipopathic peptide displaying increased Trp burial versus its Leu counterpart. Quenching experiments with a dibromo‐PC lipid indicated deeper membrane penetration of the Ile versus the Leu lipopathic peptide—a result supported by protease degradation assays where Ile peptides reconstituted into lipid bilayers were significantly more protected from the protease than the Leu peptides. Assessment of Trp blue shifts in the presence of lipid bilayers of varied lipid packing indicated that Leu/Ile peptide interactions are dependent on lipid composition. The overall results suggest that two main interactions tend to dominate Leu and Ile interactions within the membrane: (1) hydrophobic interactions between amino acid side chains and the surrounding lipid; and (2) degree of disruption of lipid–lipid packing. This “battle of giants” likely underlies the specific role(s) that Leu and Ile will play in the folding of a given membrane protein.
Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish ...specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate–polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity in Leu-block segments, promote translocon-mediated insertion.
Our meagre understanding of CFTR misfolding and its reversal by small-molecule correctors hampers the development of mechanism-based therapies of cystic fibrosis. Here we exploit a helical-hairpin ...construct-the simplest proxy of membrane-protein tertiary contacts-containing CFTR's transmembrane helices 3 and 4 and its corresponding disease phenotypic mutant V232D to gain molecular-level insights into CFTR misfolding and drug rescue by the corrector Lumacaftor. Using a single-molecule FRET approach to study hairpin conformations in lipid bilayers, we find that the wild-type hairpin is well folded, whereas the V232D mutant assumes an open conformation in bilayer thicknesses mimicking the endoplasmic reticulum. Addition of Lumacaftor reverses the aberrant opening of the mutant hairpin to restore a compact state as in the wild type. The observed membrane escape of the V232D hairpin and its reversal by Lumacaftor complement cell-based analyses of the full-length protein, thereby providing in vivo and in vitro correlates of CFTR misfolding and drug-action mechanisms.
Exercise is known to facilitate physical and emotional adjustment among women treated for breast cancer, and exercise exerts a profound effect on clinical depression. However, the effect of exercise ...on reducing clinical depression among breast cancer patients has not been demonstrated, especially among ethnic minority women who have a higher incidence of depression and higher mortality following breast cancer. First, literature is presented to assess exercise effects on depression among women with breast cancer. Second, we present the results of a randomized controlled trial assessing the effect of a structured exercise intervention on depression and exercise behavior in a multiethnic sample of women with early stage breast cancer enrolled prior to the start of adjuvant treatment. Results suggest that, in comparison to population norms, the rate of depression was higher in breast cancer patients. Analyses further showed that the intervention significantly increased self-reported exercise and reduced depression. These data suggest that the beneficial effects of exercise may extend to breast cancer patients with depression and may be initiated prior to and during cancer treatment
This description of a large cohort of patients broadens the spectrum of clinical disease phenotypes linked to
CARD11
DN mutations, including atopy, cutaneous viral and respiratory infections, ...hypogammaglobulinemia, autoimmunity, neutropenia, and lymphoma.
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