Project

Biochemical and Physiological Characterization of LEA proteins from Artemia franciscana

Goal: To elucidate the role of LEA proteins from groups 1, 3 and 6 in desiccation tolerance of encysted brine shrimp embryos.

Methods: Protein Purification, Calorimetry, Confocal Microscope, High Resolution Respirometry, Ectopic gene expression

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Michael A Menze
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The observation that some animals can survive severe dehydration in a state approaching suspended animation (anhydrobiosis) has sparked fascination since Antony van Leeuwenhoek first described it in the early 18th century for a species of bdelloid rotifers. Centuries later, we discovered a group of intrinsically disordered polypeptides, named LEA proteins, strongly implicated in increasing desiccation tolerance in plants and many anhydrobiotic animals. Recent findings demonstrate that some LEA proteins form proteinaceous droplets during water loss, which selectively partition biomolecules and may organize the cytosol into regions of biochemically distinct refugees for dehydration-sensitive biomolecules. A thorough understanding of the molecular principles governing anhydrobiosis, and the role of proteinaceous liquid–liquid phase separation in the process, is required to engineer this trait into organisms susceptible to water stress. Engineering anhydrobiosis into other organisms would revolutionize areas spanning from how we preserve medically relevant cells and tissues to securing our food supply.
Michael A Menze
added a research item
To deal with increasingly severe periods of dehydration related to global climate change, it becomes increasingly important to understand the complex strategies many organisms have developed to cope with dehydration and desiccation. While it is undisputed that late embryogenesis abundant (LEA) proteins play a key role in the tolerance of plants and many anhydrobiotic organisms to water limitation, the molecular mechanisms are not well understood. In this review we recap the current knowledge of the physiological roles of LEA proteins and discuss their potential molecular functions. As these are ultimately linked to conformational changes in the presence of binding partners, posttranslational modifications or water deprivation, we give a detailed summary of the current knowledge on the structure-function relationship of LEA proteins, including their disordered state in solution, coil-to-helix transitions, self-assembly and their recently discovered ability to undergo liquid-liquid phase separation (LLPS). We point out the promising potential of LEA proteins in biotechnological and agronomic applications and summarize recent advances. We identify the most relevant open questions and discuss major challenges in establishing a solid understanding of how these intriguing molecules accomplish their tasks as cellular sentinels at the limits of surviving water scarcity.
Brett Janis
added a research item
Group 1 (Dur-19, PF00477, LEA_5) Late Embryogenesis Abundant (LEA) proteins are present in organisms from all three domains of life, Archaea, Bacteria, and Eukarya. Surprisingly, Artemia is the only genus known to include animals that express group 1 LEA proteins in their desiccation-tolerant life-history stages. Bioinformatics analysis of circular dichroism data indicates that the group 1 LEA protein AfLEA1 is surprisingly ordered in the hydrated state and undergoes during desiccation one of the most pronounced disorder-to-order transitions described for LEA proteins from A. franciscana. The secondary structure in the hydrated state is dominated by random coils (42%) and β-sheets (35%) but converts to predominately α-helices (85%) when desiccated. Interestingly, AfLEA1 interacts with other proteins and nucleic acids, and RNA promotes liquid–liquid phase separation (LLPS) of the protein from the solvent during dehydration in vitro. Furthermore, AfLEA1 protects the enzyme lactate dehydrogenase (LDH) during desiccation but does not aid in restoring LDH activity after desiccation-induced inactivation. Ectopically expressed in D. melanogaster Kc167 cells, AfLEA1 localizes predominantly to the cytosol and increases the cytosolic viscosity during desiccation compared to untransfected control cells. Furthermore, the protein formed small biomolecular condensates in the cytoplasm of about 38% of Kc167 cells. These findings provide additional evidence for the hypothesis that the formation of biomolecular condensates to promote water stress tolerance during anhydrobiosis may be a shared feature across several groups of LEA proteins that display LLPS behaviors. Keywords: protein condensate; water stress; cryptobiosis; extremophiles; late embryogenesis abundant; LLPS
Brett Janis
added a research item
Significance Recent research has shown that intracellular proteinaceous condensates (membraneless organelles [MLOs]) are involved in various processes, ranging from Alzheimer’s disease to RNA processing, and here we demonstrate that this phenomenon governs a mechanism of anhydrobiosis. The protein Afr LEA6 is found in the desiccation-tolerant life stage of the animal extremophile Artemia franciscana, and the protein engages in two distinct molecular mechanisms to confer protection during water loss. Afr LEA6 forms MLOs that may act as protective nodes for desiccation-sensitive proteins, while a cytosolic fraction of the protein promotes structural integrity of cells during anhydrobiosis. These findings significantly advance our understanding of “life without water” and promote transformative advancements in various fields, ranging from cell preservation technology to improvement of crop desiccation tolerance.
Michael A Menze
added a research item
Late embryogenesis abundant (LEA) proteins are found in desiccation-tolerant species from all domains of life. Despite several decades of investigation, the molecular mechanisms by which LEA proteins confer desiccation tolerance are still unclear. In this study, dielectrophoresis (DEP) was used to determine the electrical properties of Drosophila melanogaster (Kc167) cells ectopically expressing LEA proteins from the anhydrobiotic brine shrimp, Artemia franciscana. Dielectrophoresis-based characterization data demonstrate that the expression of two different LEA proteins, AfrLEA3m and AfrLEA6, increases cytoplasmic conductivity of Kc167 cells to a similar extent above control values. The impact on cytoplasmic conductivity was surprising, given that the concentration of cytoplasmic ions is much higher than the concentrations of ectopically expressed proteins. The DEP data also supported previously reported data suggesting that AfrLEA3m can interact directly with membranes during water stress. This hypothesis was strengthened using scanning electron microscopy, where cells expressing AfrLEA3m were found to retain more circular morphology during desiccation, while control cells exhibited a larger variety of shapes in the desiccated state. These data demonstrate that DEP can be a powerful tool to investigate the role of LEA proteins in desiccation tolerance and may allow to characterize protein-membrane interactions in vivo, when direct observations are challenging.
Michael A Menze
added a research item
Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins (IDPs) commonly found in anhydrobiotic organisms and are frequently correlated with desiccation tolerance. Herein we report new findings on AfrLEA6, a novel group 6 LEA protein from embryos of Artemia franciscana. Assessment of secondary structure in aqueous and dried states with circular dichroism (CD) reveals 89% random coil in the aqueous state, thus supporting classification of AfrLEA6 as an IDP. Removal of water from the protein by drying or exposure to trifluoroethanol (a chemical de-solvating agent) promotes a large gain in secondary structure of AfrLEA6, predominated by α-helix and exhibiting minimal β-sheet structure. We evaluated the impact of physiological concentrations (up to 400 mM) of the disaccharide trehalose on the folding of LEA proteins in solution. CD spectra for AfrLEA2, AfrLEA3m, and AfrLEA6 are unaffected by this organic solute noted for its ability to drive protein folding. AfrLEA6 exhibits its highest concentration in vivo during embryonic diapause, drops acutely at diapause termination, and then declines during development to undetectable values at the larval stage. Maximum cellular titer of AfrLEA6 was 10-fold lower than for AfrLEA2 or AfrLEA3, both group 3 LEA proteins. Acute termination of diapause with H2O2 (a far more effective terminator than desiccation in this Great Salt Lake, UT, population) fostered a rapid 38% decrease in AfrLEA6 content of embryos. While the ultimate mechanism of diapause termination is unknown, disruption of key macromolecules could initiate physiological signaling events necessary for resumption of development and metabolism.
Michael A Menze
added 3 research items
Late Embryogenesis Abundant (LEA) proteins are a remarkable group of intrinsically disordered proteins (IDPs) that confer desiccation tolerance to plants and animals that can enter a cryptobiotic state during their life cycle. AfrLEA6 contains seed maturation domains (SMD) and is expressed in the anhydrobiotic cysts of the brine shrimp Artemia franciscana. in vitro analyses of AfrLEA6 reveal a series of protein phase transitions during desiccation. As ionic strength or molecular crowding with Ficoll-400 increases, AfrLEA6 undergoes a liquid-liquid phase separation (LLPS), forming protein droplets. AfrLEA6 droplets are also inducible by reducing the sample pH from 8.0 to 6.5 and cooling protein solutions from 25˚C to 4˚C. These conditions are notable in the context of the cysts of A. franciscana, which can naturally undergo a cytoplasmic pH shift from 7.9 to 6.5 in response to severe hypoxia. In the hydrated state, AfrLEA6 droplets exclude green fluorescent protein demonstrating that the protein droplet may be selective for inclusions of specific targets. SEM and AFM reveal that AfrLEA6 may also undergoes a phase shift to a hydrogel structure, as ionic strength and crowding increase, which is reversible upon rehydration. However, early during dehydration formed hydrogels dry into a reversible glassy state during complete desiccation. The LLPS of AfrLEA6 may confer desiccation tolerance by selectively incorporating sensitive protein targets and shielding them from desiccation induced denaturation during early drying. Any incorporated proteins may then be stabilized within a glassy compartment in the fully desiccated state and released upon rehydration (supported by NSF IOS-1659970).
Late Embryogenesis Abundant (LEA) proteins are a class of highly hydrophilic intrinsically disordered polypeptides (IDP) that are found in many plants and some anhydrobiotic animals. Over 15 distinct LEA proteins, belonging to three different classification groups (1,3 and 6), have been found in Artemia franciscana and several of these proteins have been shown to be involved in the anhydrobiotic life history stage of these Branchiopods. The exact mechanisms by which specific LEA proteins protect brine shrimp embryos during desiccation is largely unknown. To gain understanding into the possible mechanisms of protection conferred by group 1 and 6 LEA proteins, enzyme assays were utilized to investigate the effect of AfLEA1.1 and AfrLEA6 on lactate dehydrogenase (LDH) activity in lysate of Drosophila melanogaster Kc167 cells after desiccation and rehydration. Cell lysates were utilized to probe for specific interactions between LDH and LEA proteins during water-stress in a proteome system. This may closer resemble potential interaction in the cytoplasm than observed in a binary protein study with purified enzymes and a specific LEA protein. Results show that AfLEA1.1 added to purified LDH protected the enzyme during desiccation and rehydration, however, when added to cell lysate, no protection of enzymatic activity was observed after rehydration compared to LEA-free control lysates. Similarly, no protection of LDH activity by AfrLEA6 was observed when the protein was added to cell lysates before desiccation compared to LEA-free controls. It appears likely that the protection of enzymatic activity observed by AfLEA1.1 in the binary protein system might be an overestimate and LDH is not a specific target of AfLEA1.1 under physiological conditions (supported by NSF IOS-1659970).
Our understanding of protein liquid-liquid phase separation (LLPS; ‘membraneless organelles’) and its importance in a wide range of biological phenomena is rapidly growing. Unexpectedly, protein LLPS may also play a role in the desiccation- and osmotic-stress tolerance of encysted Artemia franciscana (brine shrimp) embryos. AfrLEA6 is an intrinsically disordered protein in Artemia that shares homology with seed maturation proteins (SMPs) found in some plant seeds. SMPs have been linked to the duration in which a seed remains viable in the dried state. Therefore, it was hypothesized that AfrLEA6 may play a role in sustained tolerance to water stress. This hypothesis was tested by ectopically expressing AfrLEA6 in desiccation-sensitive Drosophila melanogaster (Kc167) cells and exposing these cells to water stress. AfrLEA6 was found to increase both desiccation and osmotic-stress tolerance of Kc167 cells. Furthermore, confocal microscopy was used to image LLPS of AfrLEA6 in vivo. Staining cells with Nile Red, a lipophilic dye, suggested that AfrLEA6 causes the cytosol to interact with Nile Red like an aqueous-organic cosolvent mixture. Altered solvent properties may decrease the thermodynamic stability of unfolded proteins and reduce native protein conformational mobility yielding cytosolic wide stabilization of native proteins. Altogether, these data support the hypothesis that AfrLEA6 plays a role during water loss and indicates that AfrLEA6 significantly impacts the physicochemical properties of the cytosol. (Supported by NSF IOS-1659970.)
Michael A Menze
added a research item
This review compares the molecular strategies employed by anhydrobiotic invertebrates to survive extreme water stress. Intrinsically disordered proteins (IDPs) play a central role in desiccation tolerance in all species investigated. Various hypotheses about the functions of anhydrobiosis‐related intrinsically disordered (ARID) proteins, including late embryogenesis abundant (LEA) and tardigrade‐specific intrinsically disordered proteins, were evaluated by broad sequence characterization. A surprisingly wide range in sequence characteristics including hydropathy and the frequency and distribution of charges was discovered. Interestingly, two clusters of similar proteins were found that potentially correlate with distinct functions. This may indicate two broad groups of ARID proteins, composed of one group that folds into functional conformations during desiccation and a second group that potentially displays functions in the hydrated state. A broad range of physiochemical properties suggest that folding may be induced by factors such as hydration level, molecular crowding, and interactions with binding partners. This plasticity may be required to fine tune the ARID‐proteome response at different hydration levels during desiccation. Furthermore, the sequence properties of some LEA proteins share qualities with IDPs known to undergo liquid‐liquid phase separations during environmental challenges. This article is protected by copyright. All rights reserved
Michael A Menze
added a research item
Late embryogenesis abundant (LEA) proteins are a large group of anhydrobiosis-associated intrinsically disordered proteins (IDP), which are commonly found in plants and some animals. The brine shrimp Artemiafranciscana 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 A. franciscana 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 ArtemiaLEAome and to analyze the roles of intrinsic disorder in functioning of brine shrimp LEA proteins. We show here that A. franciscanaLEA proteins from different groups are more similar to each other than one originally expected, while functional differences among members of group 3 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, A. franciscana 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.
Michael A Menze
added a research item
Anhydrobiosis is an astounding strategy that allows certain species (both in animals and plants) to survive severe environmental conditions such as desiccation, extreme cold, or heat in the habitat. Among different molecular strategies, expression of highly hydrophilic polypeptides termed LEA proteins has been linked to the survival of plants and animals during periods of water stress such as freezing and drying. Several classification schemes for LEA proteins have been proposed and the brine shrimp, Artemia franciscana, is the only known animal that naturally expresses LEA proteins from three different classification groups (groups 1, 3, and 6). LEA proteins occur in several subcellular compartments including the cytosol and mitochondria. To investigate the biochemical properties of LEA proteins, it is important to characterize their structure. LEA proteins are intrinsically disordered in aqueous solution and the exact structure and function of these proteins is still poorly defined and understood. We hypothesized that LEA proteins will show different protective properties depending on the target enzyme (e.g. lactate dehydrogenase vs. succinate dehydrogenase). We found, that a purified group 1 LEA protein from A. franciscana (AfrLEA 1.1) helped to retain enzyme activity after desiccation of lactate dehydrogenase (LDH) and dry storage of the enzyme for 1, 3 and 7 days in the presence or absence of bovine serum albumin or trehalose. Increased concentration of purified AfrLEA 1.1, increased the percentage of LDH activity retained after desiccation. To further characterize AfrLEA 1.1, we cloned, expressed, and purified the protein in E. coli. We purified untagged AfrLEA 1.1 protein by affinity chromatography via intein mediated purification with an affinity based chitin-binding system; a novel protein purification system which utilizes the inducible self-cleavage activity of protein splicing elements to separate the target protein from the affinity tag. Furthermore, AfrLEA1.1 was expressed in Nicotiana tabacum to investigate if the protein increases drought tolerance of this model plant.
Michael A Menze
added 3 research items
The threat of desiccation for organisms inhabiting the intertidal zone occurs during emersion at low tides or when organisms are positioned in the high intertidal zone, where wetting occurs primarily by spring tides, storm waves, and spray. Drying due to evaporative water loss is the most common mechanism for dehydration, although during winter in northern temperate regions freezing can also occur, which reduces the liquid water in extracellular fluids and can lead to intracellular dehydration in multicellular organisms. Freezing tolerance has been reported and characterized for a number of intertidal invertebrates, including gastropods such as an air-breathing snail and a periwinkle, and bivalve genera including the common and ribbed mussels.
Late embryogenesis abundant (LEA) proteins are extremely hydrophilic proteins that were first identified in land plants. Intracellular accumulation is tightly correlated with acquisition of desiccation tolerance, and data support their capacity to stabilize other proteins and membranes during drying, especially in the presence of sugars like trehalose. Exciting reports now show that LEA proteins are not restricted to plants; multiple forms are expressed in desiccation-tolerant animals from at least four phyla. We evaluate here the expression, subcellular localization, biochemical properties, and potential functions of LEA proteins in animal species during water stress. LEA proteins are intrinsically unstructured in aqueous solution, but surprisingly, many assume their native conformation during drying. They are targeted to multiple cellular locations, including mitochondria, and evidence supports that LEA proteins stabilize vitrified sugar glasses thought to be important in the dried state. More in vivo experimentation will be necessary to fully unravel the multiple functional properties of these macromolecules during water stress.
Michael A Menze
added 15 research items
Main conclusion We have evaluated the endogenous expression and molecular properties of selected Group 3 LEA proteins from Artemia franciscana , and the capacity of selected Groups 1 and 3 proteins transfected into various desiccation-sensitive cell lines to improve tolerance to drying. Organisms inhabiting both aquatic and terrestrial ecosystems frequently are confronted with the problem of water loss for multiple reasons—exposure to hypersalinity, evaporative water loss, and restriction of intracellular water due to freezing of extracellular fluids. Seasonal desiccation can become severe and lead to the production of tolerant propagules and entry into the state of anhydrobiosis at various stages of the life cycle. Such is the case for gastrula-stage embryos of the brine shrimp, Artemia franciscana. Physiological and biochemical responses to desiccation are central for survival and are multifaceted. This review will evaluate the impact of multiple late embryogenesis abundant proteins originating from A. franciscana, together with the non-reducing sugar trehalose, on prevention of desiccation damage at multiple levels of biological organization. Survivorship of desiccation-sensitive cells during water stress can be improved by use of the above protective agents, coupled to metabolic preconditioning and rapid cell drying. However, obtaining long-term stability of cells in the dried state at room temperature has not been accomplished and will require continued efforts on both the physicochemical and biological fronts.
Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.
Michael A Menze
added a project goal
To elucidate the role of LEA proteins from groups 1, 3 and 6 in desiccation tolerance of encysted brine shrimp embryos.