Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Anchor Clustering for million-scale immune repertoire sequencing data.

BACKGROUND: The clustering of immune repertoire data is challenging due to the computational cost associated with a very large number of pairwise sequence comparisons. To overcome this limitation, we developed Anchor Clustering, an unsupervised clustering method designed to identify similar sequences from millions of antigen receptor gene sequences. First, a Point Packing algorithm is used to identify a set of maximally spaced anchor sequences. Then, the genetic distance of the remaining sequences to all anchor sequences is calculated and transformed into distance vectors. Finally, distance vectors are clustered using unsupervised clustering. This process is repeated iteratively until the resulting clusters are small enough so that pairwise distance comparisons can be performed. RESULTS: Our results demonstrate that Anchor Clustering is faster than existing pairwise comparison clustering methods while providing similar clustering quality. With its flexible, memory-saving strategy, Anchor Clustering is capable of clustering millions of antigen receptor gene sequences in just a few minutes. CONCLUSIONS: This method enables the meta-analysis of immune-repertoire data from different studies and could contribute to a more comprehensive understanding of the immune repertoire data space.

Isolation and molecular characterization of an FSK2-type dehydrin from Atriplex halimus.

Dehydrins form the group II LEA protein family and are known to play multiple roles in plant stress tolerance and enzyme protection. They harbor a variable number of conserved lysine rich motifs (K-segments) and may also contain three additional conserved motifs (Y-, F- and S-segments). In this work, we report the isolation and characterization of an FSK2-type dehydrin from the halophytic species Atriplex halimus, which we designate as AhDHN1. In silico analysis of the protein sequence revealed that AhDHN1 contains large number of hydrophilic residues, and is predicted to be intrinsically disordered. In addition, it has an FSK2 architecture with one F-segment, one S-segment, and two K-segments. The expression analysis showed that the AhDHN1 transcript is induced by salt and water stress treatments in the leaves of Atriplex seedlings. Moreover, circular dichroism spectrum performed on recombinant AhDHN1 showed that the dehydrin lacks any secondary structure, confirming its intrinsic disorder nature. However, there is a gain of α-helicity in the presence of membrane-like SDS micelles. In vitro assays revealed that AhDHN1 is able to effectively protect enzymatic activity of the lactate dehydrogenase against cold, heat and dehydration stresses. Our findings strongly suggest that AhDHN1 can be involved in the adaptation mechanisms of halophytes to adverse environments.

The difficulty of aligning intrinsically disordered protein sequences as assessed by conservation and phylogeny.

Intrinsically disordered proteins (IDPs) are proteins that lack a stable 3D structure but maintain a biological function. It has been frequently suggested that IDPs are difficult to align because they tend to have fewer conserved residues compared to ordered proteins, but to our knowledge this has never been directly tested. To compare the alignments of ordered proteins to IDPs, their multiple sequence alignments (MSAs) were assessed using two different methods. The first compared the similarity between MSAs produced using the same sequences but created with Clustal Omega, MAFFT, and MUSCLE. The second assessed MSAs based on how well they recapitulated the species tree. These two methods measure the "correctness" of an MSA with two different approaches; the first method measures consistency while the second measures the underlying phylogenetic signal. Proteins that contained both regions of disorder and order were analyzed along with proteins that were fully disordered and fully ordered, using nucleotide, codon and peptide sequence alignments. We observed that IDPs had less similar MSAs than ordered proteins, which is most likely linked to the lower sequence conservation in IDPs. However, comparisons of tree distances found that trees from the ordered sequence MSAs were not significantly closer to the species tree than those inferred from disordered sequence MSAs. Our results show that it is correct to say that IDPs are difficult to align on the basis of MSA consistency, but that this does not equate with alignments being of poor quality when assessed by their ability to correctly infer a species tree.

Proteins Involved in Plant Dehydration Protection: The Late Embryogenesis Abundant Family.

Plants have evolved a number of different ways to deal with different types of abiotic stresses; at the molecular level, dehydration can cause multiple forms of damage to different biomolecules [...].

The Effect of Positive Charge Distribution on the Cryoprotective Activity of Dehydrins.

Dehydrins are intrinsically disordered proteins expressed ubiquitously throughout the plant kingdom in response to desiccation. Dehydrins have been found to have a cryoprotective effect on lactate dehydrogenase (LDH) in vitro, which is in large part influenced by their hydrodynamic radius rather than the order of the amino acids within the sequence (alternatively, this may be a sequence specific effect). However, it seems that a different mechanism may underpin the cryoprotection that they confer to the cold-labile yeast frataxin homolog-1 (Yfh1). Circular dichroism spectroscopy (CD) was used to assess the degree of helicity of Yfh1 at 1 °C, both alone and in the presence of several dehydrin constructs. Three constructs were compared to the wild type: YSK2-K→R (lysine residues substituted with arginine), YSK2-Neutral (locally neutralized charge), and YSK2-SpaceK (evenly distributed positive charge). The results show that sequence rearrangements and minor substitutions have little impact on the ability of the dehydrin to preserve LDH activity. However, when the positive charge of the dehydrin is locally neutralized or evenly distributed, the dehydrin becomes less efficient at promoting structure in Yfh1 at low temperatures. This suggests that a stabilizing, charge-based interaction occurs between dehydrins and Yfh1. Dehydrins are intrinsically disordered proteins, expressed by certain organisms to improve desiccation tolerance. These proteins are thought to serve many cellular roles, such as the stabilization of membranes, DNA, and proteins. However, the molecular mechanisms underlying the function of dehydrins are not well understood. Here, we examine the importance of positive charges in dehydrin sequences by making substitutions and comparing their effects in the cryoprotection of two different proteins.

LEAfing through literature: late embryogenesis abundant proteins coming of age-achievements and perspectives.

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 summarize 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, post-translational modifications, or water deprivation, we provide a detailed summary of 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. 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.

Physiological, Structural, and Functional Insights Into the Cryoprotection of Membranes by the Dehydrins.

Plants can be exposed to cold temperatures and have therefore evolved several mechanisms to prevent damage caused by freezing. One of the most important targets are membranes, which are particularly susceptible to cold damage. To protect against such abiotic stresses, plants express a family of proteins known as late embryogenesis abundant (LEA) proteins. Many LEA proteins are intrinsically disordered, that is, they do not contain stable secondary or tertiary structures alone in solution. These proteins have been shown in a number of studies to protect plants from damage caused by cold, drought, salinity, and osmotic stress. In this family, the most studied proteins are the type II LEA proteins, better known as dehydrins (dehydration-induced proteins). Many physiological studies have shown that dehydrins are often located near the membrane during abiotic stress and that the expression of dehydrins helps to prevent the formation of oxidation-modified lipids and reduce the amount of electrolyte leakage, two hallmarks of damaged membranes. One of the earliest biophysical clues that dehydrins are involved in membrane cryoprotection came from in vitro studies that demonstrated a binding interaction between the protein and membranes. Subsequent work has shown that one conserved motif, known as K-segments, is involved in binding, while recent studies have used NMR to explore the residue specific structure of dehydrins when bound to membranes. The biophysical techniques also provide insight into the mechanism by which dehydrins protect the membrane from cold stress, which appears to mainly involve the lowering of the transition temperature.

The Disordered Dehydrin and Its Role in Plant Protection: A Biochemical Perspective.

Dehydrins are intrinsically disordered proteins composed of several well conserved sequence motifs known as the Y-, S-, F-, and K-segments, the latter of which is a defining feature of all dehydrins. These segments are interspersed by regions of low sequence conservation and are organized modularly, which results in seven different architectures: Kn, SKn, YnSKn, YnKn, KnS, FnK and FnSKn. Dehydrins are expressed ubiquitously throughout the plant kingdom during periods of low intracellular water content, and are capable of improving desiccation tolerance in plants. In vitro evidence of dehydrins shows that they are involved in the protection of membranes, proteins and DNA from abiotic stresses. However, the molecular mechanisms by which these actions are achieved are as of yet somewhat unclear. With regards to macromolecule cryoprotection, there is evidence to suggest that a molecular shield-like protective effect is primarily influenced by the hydrodynamic radius of the dehydrin and to a lesser extent by the charge and hydrophobicity. The interaction between dehydrins and membranes is thought to be a surface-level, charge-based interaction that may help to lower the transition temperature, allowing membranes to maintain fluidity at low temperatures and preventing membrane fusion. In addition, dehydrins are able to protect DNA from damage, showing that these abiotic stress protection proteins have multiple roles.

The Halophyte Dehydrin Sequence Landscape.

Dehydrins (DHNs) belong to the LEA (late embryogenesis abundant) family group II, that comprise four conserved motifs (the Y-, S-, F-, and K-segments) and are known to play a multifunctional role in plant stress tolerance. Based on the presence and order of these segments, dehydrins are divided into six subclasses: YnSKn, FnSKn, YnKn, SKn, Kn, and KnS. DHNs are rarely studied in halophytes, and their contribution to the mechanisms developed by these plants to survive in extreme conditions remains unknown. In this work, we carried out multiple genomic analyses of the conservation of halophytic DHN sequences to discover new segments, and examine their architectures, while comparing them with their orthologs in glycophytic plants. We performed an in silico analysis on 86 DHN sequences from 10 halophytic genomes. The phylogenetic tree showed that there are different distributions of the architectures among the different species, and that FSKn is the only architecture present in every plant studied. It was found that K-, F-, Y-, and S-segments are highly conserved in halophytes and glycophytes with a few modifications, mainly involving charged amino acids. Finally, expression data collected for three halophytic species (Puccinillia tenuiflora, Eutrema salsugenium, and Hordeum marinum) revealed that many DHNs are upregulated by salt stress, and the intensity of this upregulation depends on the DHN architecture.

Phosphorylation-dependent control of Activity-regulated cytoskeleton-associated protein (Arc) protein by TNIK.

Activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, and been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arc's subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15077.

The in vitro structure and functions of the disordered late embryogenesis abundant three proteins.

Late embryogenesis abundant (LEA) proteins are produced during seed embryogenesis and in vegetative tissue in response to various abiotic stressors. A correlation has been established between LEA expression and stress tolerance, yet their precise biochemical mechanism remains elusive. LEA proteins are very rich in hydrophilic amino acids, and they have been found to be intrinsically disordered proteins (IDPs) in vitro. Here, we perform biochemical and structural analyses of the four LEA3 proteins from Arabidopsis thaliana (AtLEA3). We show that the LEA3 proteins are disordered in solution but have regions with propensity for order. All LEA3 proteins were effective cryoprotectants of LDH in the freeze/thaw assays, while only one member, AtLEA3-4, was shown to bind Cu2+ and Fe3+ ions with micromolar affinity. As well, only AtLEA3-4 showed binding and a gain in α-helicity in the presence of the membrane mimic dodecylphosphocholine (DPC). We explored this interaction in greater detail using 15 N-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance, and demonstrate that two sets of conserved motifs present in AtLEA3-4 are involved in the interaction with the DPC micelles, which themselves gain α-helical structure.

Conserved sequence motifs in the abiotic stress response protein late embryogenesis abundant 3.

LEA3 proteins, a family of abiotic stress proteins, are defined by the presence of a tryptophan-containing motif, which we name the W-motif. We use Pfam LEA3 sequences to search the Phytozome database to create a W-motif definition and a LEA3 sequence dataset. A comprehensive analysis of these sequences revealed four N-terminal motifs, as well as two previously undiscovered C-terminal motifs that contain conserved acidic and hydrophobic residues. The general architecture of the LEA3 sequences consisted of an N-terminal motif with a potential mitochondrial transport signal and the twin-arginine motif cut-site, followed by a W-motif and often a C-terminal motif. Analysis of species distribution of the motifs showed that one architecture was found exclusively in Commelinids, while two were distributed fairly evenly over all species. The physiochemical properties of the different architectures showed clustering in a relatively narrow range compared to the previously studied dehydrins. The evolutionary analysis revealed that the different sequences grouped into clades based on architecture, and that there appear to be at least two distinct groups of LEA3 proteins based on their architectures and physiochemical properties. The presence of LEA3 proteins in non-vascular plants but their absence in algae suggests that LEA3 may have arisen in the evolution of land plants.

Expression and Purification of an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) describe a group of proteins that do not have a regular tertiary structure and typically have very little ordered secondary structure. Despite not following the biochemical dogma of "structure determines function" and "function determines structure," IDPs have been identified as having numerous biological functions. We describe here the steps to express and purify the intrinsically disordered stress response protein, Late embryogenesis abundant protein 3-2 from Arabidopsis thaliana (AtLEA 3-2), with 15N and 13C isotopes in E. coli, although the protocol can be adapted for any IDP with or without isotopic labeling. The atlea 3-2 gene has been cloned into the pET-SUMO vector that in addition to the SUMO portion encodes an N-terminal hexahistidine sequence (His-tag). This vector allows for the SUMO-AtLEA 3-2 fusion protein to be purified using Ni-affinity chromatography and, through the use of ubiquitin-like-specific protease 1 (Ulp1, a SUMO protease), results in an AtLEA 3-2 with a native N-terminus. We also describe the expression and purification of Ulp1 itself.

Effect of in vitro cold acclimation of Deschampsia antarctica on the accumulation of proteins with antifreeze activity.

Deschampsia antarctica has managed to colonize the maritime Antarctic. One of the main factors associated with its tolerance to low temperatures is the presence of apoplastic proteins with antifreeze activity. This work focuses on the effect of cold acclimation of D. antarctica on the accumulation of apoplastic proteins with antifreeze activity. Antifreeze proteins present in apoplastic extracts were purified by ice affinity purification, and their identity was determined by protein sequencing. D. antarctica plants were subjected to 22 days of cold acclimation at 4 °C. The highest content of apoplastic proteins with antifreeze activity was obtained at between 12 and 16 days of acclimation. Protein sequencing allowed their identification with >95% probability. Percentage coverage was 74% with D. antarctica ice recrystallization inhibition protein 1 (DaIRIP1) and 55% with DaIRIP3. Cold acclimation of D. antarctica improved the yield of apoplastic proteins, and resulted in an increase in the antifreeze activity of apoplastic extracts. An in silico analysis suggested that the fluctuations presented by the three-dimensional structures of DaIRIPs help to explain the presence of certain DaIRIPs in apoplastic extracts under the cold acclimation conditions evaluated.

Binding of a Vitis riparia dehydrin to DNA.

Plants must protect themselves from abiotic stresses such as drought, cold, and high salinity. The common thread of all three stresses is that they cause dehydration, which in turn promotes the formation of reactive oxygen species (ROS). Dehydrin proteins (dehydrins) are a large family of proteins that have been identified in nearly all land plants, and whose presence is correlated with plant protection from abiotic stresses. Several dehydrin studies have shown that some dehydrins localize to the nucleus, as well as the cytoplasm, but a functional role for nuclear dehydrins has not yet been determined. We show here that the Vitis riparia dehydrin VrDHN1 localizes to the nucleus and is able to bind to DNA to protect it from damage caused by hydrogen peroxide, an ROS source. We also show that the binding to DNA is not DNA-sequence specific, suggesting that the protein is able to protect any exposed DNA without interfering with its normal function. NMR studies show that the binding is largely driven by the lysine-rich nature of dehydrins located in the conserved K-segments. Unlike other, previously studied dehydrins, VrDHN1 binding to DNA is not enhanced through the presence of metals. Lastly, we demonstrate that the Y-segment does not bind ATP, as has long been proposed.

Sequence composition versus sequence order in the cryoprotective function of an intrinsically disordered stress-response protein.

Intrinsically disordered stress proteins have been shown to act as chaperones, protecting proteins from damage caused by stresses such as freezing and thawing. Dehydration proteins (dehydrins) are intrinsically disordered stress proteins that are found in almost all land plants. They consist of a variable number of the short, semi-conserved, Y-, S-, and K-segments, with longer stretches of poorly conserved sequences in between. Previous studies have provided conflicting views on the details of the dehydrin cryoprotective mechanism of enzymes. Experiments with polyethylene glycol (PEG) have shown that PEG cryoprotective efficiency is the same as dehydrins of the same hydrodynamic radius, suggesting that the protein's disordered and polar nature is important, rather than the specific order of the residues. To further elucidate the mechanism, we created scrambled variants of the wild grape dehydrins K2 and YSK2 and tested their ability to protect lactate dehydrogenase and yeast frataxin homolog-1 from freeze/thaw damage. The results show that for preventing aggregation, it is the sequence composition and the size of the dehydrin that is the most important factor in protection, while for freeze/thaw damage causing loss of secondary structure, it is the sequence composition that is most significant.