Potential functions of LEA proteins from the brine shrimp <i>Artemia franciscana</i> – anhydrobiosis meets bioinformatics

<p>Late embryogenesis abundant (LEA) proteins are a large group of anhydrobiosis-associated intrinsically disordered proteins, which are commonly found in plants and some animals. The brine shrimp <i>Artemia franciscana</i> is the only known animal that expresses LEA proteins from three, and not only one, different groups in its anhydrobiotic life stage. The reason for the higher complexity in the <i>A. franciscana</i> LEA proteome (LEAome), compared with other anhydrobiotic animals, remains mostly unknown. To address this issue, we have employed a suite of bioinformatics tools to evaluate the disorder status of the <i>Artemia</i> LEAome and to analyze the roles of intrinsic disorder in functioning of brine shrimp LEA proteins. We show here that <i>A. franciscana</i> LEA proteins from different groups are more similar to each other than one originally expected, while functional differences among members of group three are possibly larger than commonly anticipated. Our data show that although these proteins are characterized by a large variety of forms and possible functions, as a general strategy, <i>A. franciscana</i> utilizes glassy matrix forming LEAs concurrently with proteins that more readily interact with binding partners. It is likely that the function(s) of both types, the matrix-forming and partner-binding LEA proteins, are regulated by changing water availability during desiccation.</p

Quantification of intracellular trehalose in wild-type CHO cells and CHO-TRET1 cells.

<p>Cells were incubated in fully complemented cell culture medium containing 400 mM trehalose for 4 hours (<i>n</i> = 3, ± SD).</p

Survival of CHO-TRET1 cells spin-dried in solutions with or without trehalose, then stored in LN₂ for 1 h, and finally rehydrated.

<p>(A) Membrane integrity of spin-dried cells stored in LN₂ for 1 h and 45 min after thawing and rehydration (B) Micrograph of the spin-dried cells after thawing and rehydration. (C) Growth of spin-dried cells after thawing and rehydration. The values were normalized to the initial cell count (<i>n</i> = 10, ± SD).</p

Glass transition temperature of the dried spinning solution (1.8 M trehalose, 10 mM KCl, 5 mM glucose, 20 mM HEPES, 120 mM choline chloride, pH 7.4) using FTIR technique.

<p>The figure indicates a characteristic peak-position of ν-OH stretch plotted against decreasing temperature.</p

Basic configuration of the spin-drying apparatus.

<p>The cells were grown on glass cover slips prior to the spin-drying. During spin-drying, the glass cover slip was held in place by a vacuum chuck.</p

Survival of CHO-TRET1 cells spin-dried in buffers with or without trehalose and rehydrated immediately following desiccation.

<p>(A) Membrane integrity of spin-dried cells 45 min after rehydration (B) Micrograph of the cell samples after spin drying and rehydration. (C) Growth of cells after spin-drying and rehydration. The values were normalized to the initial cell count (<i>n</i> = 10, ± SD).</p

FTIR analysis of the dried film obtained after spin drying of sample buffer (1.8 M trehalose, 10 mM KCl, 5 mM glucose, 20 mM HEPES, 120 mM choline chloride, pH 7.4).

<p>The ratio of I₁₆₅₀/I₁₁₅₀ was used to quantify local water contents.</p

Identification of Disulfide Bond Formation between MitoNEET and Glutamate Dehydrogenase 1

MitoNEET is a protein that was identified as a drug target for diabetes, but its cellular function as well as its role in diabetes remains elusive. Protein pull-down experiments identified glutamate dehydrogenase 1 (GDH1) as a potential binding partner. GDH1 is a key metabolic enzyme with emerging roles in insulin regulation. MitoNEET forms a covalent complex with GDH1 through disulfide bond formation and acts as an activator. Proteomic analysis identified the specific cysteine residues that participate in the disulfide bond. This is the first report that effectively links mitoNEET to activation of the insulin regulator GDH1