Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape

2011 ◽  
Vol 435 (2) ◽  
pp. 475-487 ◽  
Author(s):  
René Bartz ◽  
Haihong Fan ◽  
Jingtao Zhang ◽  
Nathalie Innocent ◽  
Craig Cherrin ◽  
...  
Keyword(s):  
Sirna Delivery ◽  
Specific Interaction ◽  
Direct Interaction ◽  
Helical Structure ◽  
Endosomal Escape ◽  
Titration Calorimetry ◽  
Cell Fractionation ◽  
Amphipathic Peptide ◽  
Ph Dependent

Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.

scholarly journals Serum Stability and Physicochemical Characterization of a Novel Amphipathic Peptide C6M1 for SiRNA Delivery

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e97797 ◽  
Author(s):  
Mousa Jafari ◽  
Wen Xu ◽  
Ran Pan ◽  
Chad M. Sweeting ◽  
Desiree Nedra Karunaratne ◽  
...  
Keyword(s):  
Sirna Delivery ◽  
Physicochemical Characterization ◽  
Amphipathic Peptide ◽  
Serum Stability

Structural characterization of the model amphipathic peptide Ac-LKKLLKLLKKLLKL-NH2 in aqueous solution and with 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoroisopropanol

2013 ◽  
Vol 91 (6) ◽  
pp. 406-413 ◽  
Author(s):  
Garry W. Buchko ◽  
Avijita Jain ◽  
Matthew L. Reback ◽  
Wendy J. Shaw
Keyword(s):  
Aqueous Solution ◽  
Self Assembly ◽  
Helical Structure ◽  
Cd Spectroscopy ◽  
Size Exclusion ◽  
Amphipathic Peptide ◽  
Amphipathic Peptides ◽  
Fluorinated Alcohols ◽  
Dilute Aqueous Solution

Short-chain amphipathic peptides are promising components in the new generation of engineered biomaterials. The model 14-residue leucine–lysine peptide Ac-LKKLLKLLKKLLKL-NH2 (LKα) is one such amphipathic peptide. In dilute aqueous solution (<0.05 mmol/L), it was previously proposed, using CD spectroscopic data, that LKα existed in a cooperative monomeric (unstructured) – tetrameric (α-helical) equilibrium that shifted towards the tetramer at high NaCl and peptide concentrations. Here, at similar peptide concentrations, CD spectroscopy shows that LKα readily adopts α-helical structure in the presence of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) with maximal helical character in 20% TFE and ∼10% HFIP (v/v). The helical character in fluorinated alcohols suggested by the CD data at low peptide concentrations (0.06 mmol/L) is corroborated at high peptide concentrations (1.5 mmol/L) by NMR NOE data that also show that 1.5 mmol/L LKα is helical in 100% water. Size exclusion chromatography and estimations of rotational correlation times (τc) showed that the self-assembled LKα complexes contained three to five peptides. Removing the N-terminal acetyl group prevents LKα from forming helices and self-associating at high NaCl and peptide concentrations. This more detailed characterization of the structural and physical properties of LKα over a greater range of peptide concentrations and in the presence of fluorinated alcohols will assist the design of biomaterials containing amphipathic peptides and guide the ability to control self-assembly.

scholarly journals A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1

2021 ◽  
Vol 22 (14) ◽  
pp. 7390
Author(s):  
Nicole Wesch ◽  
Frank Löhr ◽  
Natalia Rogova ◽  
Volker Dötsch ◽  
Vladimir V. Rogov
Keyword(s):  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Biochemical Characterization ◽  
Effective Interaction ◽  
Regulatory Region ◽  
Titration Calorimetry ◽  
Enzymatic Reactions ◽  
Cellular Processes ◽  
Mechanism Of Interaction

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

scholarly journals Linearized esculentin-2EM shows pH dependent antibacterial activity with an alkaline optimum

2021 ◽  
Author(s):  
Erum Malik ◽  
David A. Phoenix ◽  
Timothy J. Snape ◽  
Frederick Harris ◽  
Jaipaul Singh ◽  
...  
Keyword(s):  
Helical Structure ◽  
Food Preservation ◽  
Forming Process ◽  
Gram Positive ◽  
Gram Negative ◽  
Bacterial Membranes ◽  
Gram Positive Bacteria ◽  
Alkaline Conditions ◽  
Ph Dependent ◽  
Gram Negative Organisms

AbstractHere the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5–26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3–5.1 mN m−1) and lyse (↑ 15.1–32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1–23) in the N → C direction, with −  < µH > increasing overall from circa − 0.8 to − 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.

scholarly journals Thermodynamic characterization of calcium-milk protein interaction by isothermal titration calorimetry

2009 ◽  
Vol 89 (3-4) ◽  
pp. 257-267 ◽  
Author(s):  
Latha-Selvi Canabady-Rochelle ◽  
Christian Sanchez ◽  
Michel Mellema ◽  
Sylvie Banon
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Protein Interaction ◽  
Milk Protein ◽  
Titration Calorimetry ◽  
Thermodynamic Characterization

scholarly journals Crystal structure of thermostable alkylsulfatase SdsAP from Pseudomonas sp. S9

2017 ◽  
Vol 37 (3) ◽  
Author(s):  
Lifang Sun ◽  
Pu Chen ◽  
Yintao Su ◽  
Zhixiong Cai ◽  
Lingwei Ruan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Sodium Dodecyl ◽  
Titration Calorimetry ◽  
Pseudomonas Sp ◽  
Sterol Carrier Protein ◽  
Dimerization Domain ◽  
Alkyl Chains ◽  
Hydrolysis Of

A novel alkylsulfatase from bacterium Pseudomonas sp. S9 (SdsAP) was identified as a thermostable alkylsulfatases (type III), which could hydrolyze the primary alkyl sulfate such as sodium dodecyl sulfate (SDS). Thus, it has a potential application of SDS biodegradation. The crystal structure of SdsAP has been solved to a resolution of 1.76 Å and reveals that SdsAP contains the characteristic metallo-β-lactamase-like fold domain, dimerization domain, and C-terminal sterol carrier protein type 2 (SCP-2)-like fold domain. Kinetic characterization of SdsAP to SDS by isothermal titration calorimetry (ITC) and enzymatic activity assays of constructed mutants demonstrate that Y246 and G263 are important residues for its preference for the hydrolysis of ‘primary alkyl’ chains, confirming that SdsAP is a primary alkylsulfatase.

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