Hand 2015 Abstract MiP2015

From Bioblast
Liposomes simulating the compositions of outer and inner mitochondrial membranes are protected during desiccation by LEA Proteins from Artemia franciscana.


Hand SC, Moore DS, Hansen R (2015)

Event: MiP2015

Intracellular accumulation of Late Embryogenesis Abundant (LEA) proteins [1] and the disaccharide trehalose [2] is associated with cellular desiccation tolerance in a number of animal species. LEA proteins are a family of intrinsically disordered proteins that are unstructured in solution and adopt secondary structure as water is removed. During drying, LEA proteins protect target enzymes, prevent protein aggregation, and some may form amphipathic alpha-helices capable of interacting with lipid bilayers. Targeting of LEA proteins to different compartments within the cell emphasizes the necessity of protecting organelles from water stress-induced damage. It has been hypothesized that a given LEA protein may preferentially stabilize membranes of a particular lipid composition based on the protein’s subcellular location. Here we evaluate the protection of liposomes in the dried state by two LEA proteins from Artemia franciscana, by the sugar trehalose, and by LEA protein and trehalose in combination. Using both a cytoplasmic-localized (AfrLEA2) and a mitochondrial-targeted LEA protein (AfrLEA3m) [3,4] allowed us to test the above hypothesis.

Small unilamellar liposomes with compositions that mimicked the inner mitochondrial membrane with cardiolipin (IMM), outer mitochondrial membrane (OMM), and the inner leaflet of the plasma membrane (ILPM) were prepared with a hand held mini extruder. Desiccation-induced damage to liposomes was assessed by carboxyfluorescein leakage after air drying overnight and rehydration. Recombinant AfrLEA3m and AfrLEA2 were purified as described previously [3]. To compare the impact of LEA proteins to a negative control (i.e., a protein predicted to be non-stabilizing), liposomes were also dried with lysozyme at identical protein:lipid mass ratios. Primary amino acid sequences of AfrLEA3m and AfrLEA2 were determined from our existing cDNA library for A. franciscana and used for molecular modeling.

Both LEA proteins were able to offset damage during drying of liposomes that mimicked the lipid compositions of the IMM, OMM and ILPM (Fig. 1). Thus liposome stabilization by AfrLEA3m or AfrLEA2 was not dependent on lipid composition, provided physiological amounts of bilayer and non-bilayer-forming lipids were present (liposomes with a non-biological composition of 100% phosphatidylcholine were not protected by either protein). Stabilization by LEA proteins was significantly greater than that afforded by lysozyme for all membranes except 100 % PC liposomes (2-way ANOVA, p ≀ 0.05, n= 6). Additive protection by LEA proteins plus trehalose was dependent on the lipid composition of the target membrane. Consistent with the ability to stabilize lipid bilayers, molecular modeling of the secondary structures for AfrLEA2 and AfrLEA3m revealed bands of charged amino acids similar to other amphipathic proteins that interact directly with membranes (Fig. 2). Amino acids of positive and negative charge align in parallel bands, with acidic (negative) residues flanked to either side by basic (positive) residues. Such organization has been proposed to allow directly interact with the headgroups of lipid bilayers in the case of a plant LEA protein.

β€’ O2k-Network Lab: US LA Baton Rouge Hand SC

Labels: MiParea: mt-Membrane 

Event: B1, Oral  MiP2015 


Dept Biol Sc, Louisiana State Univ, Baton Rouge, USA. - [email protected].

Abstract continued

LEA proteins and trehalose stabilize liposomes that mimicked biological membranes when desiccated. Neither the cytoplasmic-localized AfrLEA2 nor the mitochondrial-targeted AfrLEA3m exhibits preferential protection of one compositional type of liposome over another. Matrix-resident AfrLEA3m is not more proficient at stabilizing IMM-like liposomes that contain cardiolipin than is AfrLEA2. When trehalose and LEA proteins are used in combination, IMM-like liposomes and ILPM liposomes are protected to a significantly greater degree than when dried with either protectant alone. Modeling of AfrLEA2 and AfrLEA3m as Ξ±-helices shows arrangements of charged amino acids that are consistent with other amphipathic proteins capable of direct interaction with lipid bilayers.


MiP2015 Hand Figure.JPG

Figure 1. Carboxyfluorescein leakage from liposomes dried overnight and rehydrated in the presence of LEA proteins or a control protein (lysozyme). Data represents the mean Β± SD of n = 6 samples.

Figure 2. (A) Two views of AfrLEA3m modeled as an Ξ±-helix. Charged amino acids are depicted in red (acidic: D or E) or blue (basic: H, K, or R). Hydrophobic residues (A, G, I, L, M, V, or W) are colored gray and hydrophilic residues (N, Q, S, T, or Y) are depicted as yellow. (B) The Ξ±-helical backbone (white) is depicted with the charged residues (colored as above) between positions 45-219. (C) End-on view of residues 149-240 with only the charged amino acids visible.

References and acknowledgements

  1. Hand SC, Menze MA (2015) Molecular approaches for improving desiccation tolerance: insights from the brine shrimp Artemia franciscana. Planta 242:379-88.
  2. Abazari A, Chakraborty N, Hand SC, Aksan A, Toner M (2014) A Raman micro-spectroscopy study of water and trehalose in spin-dried cells. Biophysical J. 107:2253-62.
  3. Boswell LC, Menze MA, Hand SC (2014) Group 3 LEA proteins from embryos of Artemia franciscana: Structural properties and protective abilities during desiccation. Physiol Biochem Zool 87:640-51.
  4. Boswell LC, Hand SC (2014) Intracellular localization of group 3 LEA proteins in embryos of Artemia franciscana. Tissue and Cell 46:514-19.

Supported by National Science Foundation grants IOS-0920254 and IOS-1457061/IOS-1456809

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