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.

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.

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.

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.

Testing the rogue taxa hypothesis for clustering instability.

There have been longstanding concerns about the stability of hierarchical clustering. A suggested explanation for this instability is the presence of "rogue taxa", i.e. taxa whose removal from a data set can apparently restore stability. In this study, the rogue taxa hypothesis is tested by partitioning a large data set into many smaller ones and checking for rogue behavior. The checking was performed with a standard hierarchical clustering algorithm and with a novel algorithm designed to have greater stability. It was found that rogue taxa cannot reasonably be said to exist because the status of being a rogue taxon depends on the data partition in which the taxon is embedded. In addition to the choice of data used, the choice of algorithm and algorithm parameters can have a large effect on the degree to which a taxon appears rogue. Instability in hierarchical clustering can be increased by problematic data points, but the status of data points being problematic depends not on their biological antecedents, but on their position in the local geometry of the data. The results of this study strongly suggest that instability in traditional hierarchical clustering routines is primarily a problem with the algorithm design.

Effect of an Intrinsically Disordered Plant Stress Protein on the Properties of Water.

Dehydrins are plant proteins that are able to protect plants from various forms of dehydrative stress such as drought, cold, and high salinity. Dehydrins can prevent enzymes from losing activity after freeze/thaw treatments. Previous studies had suggested that the dehydrins function by a molecular shield effect, essentially preventing a denatured enzyme from aggregating with another enzyme. Therefore, the larger the dehydrin, the larger the shield and theoretically the more effective the protection. Although this relationship holds for smaller dehydrins, it fails to explain why larger dehydrins are less efficient than would be predicted from their size. Using solvatochromic dyes to probe the solvent features of water, we first confirm that the dehydrins do not bind the dyes, which would interfere with interpretation of the data. We then show that the dehydrins have an effect on three solvent properties of water (dipolarity/polarizability, hydrogen-bond donor acidity and hydrogen-bond acceptor basicity), which can contribute to the protective mechanism of these proteins. Interpretation of these data suggests that although polyethylene glycol and dehydrins have similar protective effects, dehydrins may more efficiently modify the hydrogen-bonding ability of bulk water to prevent enzyme denaturation. This possibly explains why dehydrins recover slightly more enzyme activity than polyethylene glycol.

Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere.

Genome analyses are being used to characterize plant growth-promoting (PGP) bacteria living in different plant compartiments. In this context, we have recently isolated bacteria from the phyllosphere of an Antarctic plant (Deschampsia antarctica) showing ice recrystallization inhibition (IRI), an activity related to the presence of antifreeze proteins (AFPs). In this study, the draft genomes of six phyllospheric bacteria showing IRI activity were sequenced and annotated according to their functional gene categories. Genome sizes ranged from 5.6 to 6.3 Mbp, and based on sequence analysis of the 16S rRNA genes, five strains were identified as Pseudomonas and one as Janthinobacterium. Interestingly, most strains showed genes associated with PGP traits, such as nutrient uptake (ammonia assimilation, nitrogen fixing, phosphatases, and organic acid production), bioactive metabolites (indole acetic acid and 1-aminocyclopropane-1-carboxylate deaminase), and antimicrobial compounds (hydrogen cyanide and pyoverdine). In relation with IRI activity, a search of putative AFPs using current bioinformatic tools was also carried out. Despite that genes associated with reported AFPs were not found in these genomes, genes connected to ice-nucleation proteins (InaA) were found in all Pseudomonas strains, but not in the Janthinobacterium strain.

Cold tolerance mechanisms of two arthropods from the Andean Range of Central Chile: Agathemera crassa (Insecta: Agathemeridae) and Euathlus condorito (Arachnida: Theraphosidae).

Two strategies have been described for cold tolerance in arthropods: (1) freeze-tolerant organisms, which can survive the formation of ice crystals and (2) freeze-avoidant organisms, which prevent the ice crystal formation by super cooling their internal fluids. We studied two arthropods from the Andean Range in central Chile (2400 m a.s.l.), the stick insect Agathemera crassa commonly named as "Chinchemolle", and the tarantula spider Euathlus condorito commonly named as "Araña pollito", in order to evaluate how they respond to low temperatures at the physiological and molecular levels. We sampled the soil temperature during one year to track the temperature changes that these organisms must overcome. We found minimum temperatures around -6 °C in autumn, while the temperature were stable at 0 °C in winter due to the snow. The average field-cooling rate was 0.01 ±â€¯0.006 °C min-1. For both arthropods we determined the super cooling point (SCP) at a cooling rate of 1 °C min-1 and its subsequent survival, finding that A. crassa is a freezing tolerant organism with a SCP of -3.8 ±â€¯1.8 °C and 100% survival, while E. condorito is a freezing avoidant organism with a SCP of -3.0 ±â€¯1.3 °C and 0% survival. The SCP and survival were not affected by the season in which individuals were collected, the SCP was significantly affected by the cooling rate of the experiment. Both species had low molecular weight cryoprotective in their hemolymph that could explain their cold-tolerance behavior. Glucose, glycerol, and trehalose were found in A. crassa's hemolymph, only glucose and glycerol were found in E. condorito's. We analyzed the hemolymph proteins and found no seasonal differences in composition for either species and also we detected protein antifreeze activity in the hemolymph from both arthropods.

CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure.

The ability to survive and reproduce after cold exposure is important in all kingdoms of life. However, even in a sophisticated genetic model system like Drosophila melanogaster, few genes have been identified as functioning in cold tolerance. The accumulation of the Frost (Fst) gene transcript increases after cold exposure, making it a good candidate for a gene that has a role in cold tolerance. Despite extensive RNAi knockdown analysis, no role in cold tolerance has been assigned to Fst CRISPR is an effective technique for completely knocking down genes, and is less likely to produce off-target effects than GAL4-UAS RNAi systems. We have used CRISPR-mediated homologous recombination to generate Fst-null alleles, and these Fst alleles uncovered a requirement for FST protein in maintaining female fecundity following cold exposure. However, FST does not have a direct role in survival following cold exposure. FST mRNA accumulates in the Malpighian tubules, and the FST protein is a highly disordered protein with a putative signal peptide for export from the cell. Future work is needed to determine whether FST is exported from the Malpighian tubules and directly interacts with female reproductive tissues post-cold exposure, or whether it is required for other repair/recovery functions that indirectly alter energy allocation to reproduction.

Structure of an Intrinsically Disordered Stress Protein Alone and Bound to a Membrane Surface.

Dehydrins are a group of intrinsically disordered proteins that protect plants from damage caused by drought, cold, and high salinity. Like other intrinsically disordered proteins, dehydrins can gain structure when bound to a ligand. Previous studies have shown that dehydrins are able to protect liposomes from cold damage, but the interactions that drive membrane binding and the detailed structure of the bound and unbound forms are not known. We use an ensemble-structure approach to generate models of a dehydrin known as K2 in the presence and absence of sodium dodecyl sulfate micelles, and we docked the bound structure to the micelle. The collection of residual dipolar coupling data, amide protection factors, and paramagnetic relaxation enhancement distances, in combination with chemical shifts and relaxation measurements, allows for determining plausible structures that are not otherwise visible in time-averaged structural data. The results show that in the bound structure, the conserved lysines are important for membrane binding, whereas the flanking hydrophobic residues play a lesser role. The unbound structure shows a high level of disorder and an extended structure. We propose that the structural differences between bound and unbound forms allow dehydrins to act as molecular shields in their unbound state and as membrane protectants in their bound state. Unlike α-synuclein, the significant gain of α-helicity in K2 at low concentrations of sodium dodecyl sulfate is not due to a decrease in the critical micelle concentration. The study provides structural insight into how a disordered protein can interact with a membrane surface.

Structural and Functional Insights into the Cryoprotection of Membranes by the Intrinsically Disordered Dehydrins.

Dehydration can be due to desiccation caused by a lack of environmental water or to freezing caused by a lack of liquid water. Plants have evolved a large family of proteins called LEA (late embryogenesis abundant) proteins, which include the intrinsically disordered dehydrin (dehydration protein) family, to combat these abiotic stresses. Although transcription and translation studies have shown a correlation between dehydration stress and the presence of dehydrins, the biochemical mechanisms have remained somewhat elusive. We examine here the effect and structure of a small model dehydrin (Vitis riparia K2) on the protection of membranes from freeze-thaw stress. This protein is able to bind to liposomes containing phosphatidic acid and protect the liposomes from fusing after freeze-thaw treatment. The presence of K2 did not measurably affect liposome surface accessibility or lipid mobility but did lower its membrane transition temperature by 3 °C. Using sodium dodecyl sulfate as a membrane model, we examined the NMR structure of K2 in the presence and absence of the micelle. Biochemical and NMR experiments show that the conserved, lysine-rich segments are involved in the binding of the dehydrin to a membrane, whereas the poorly conserved φ segments play no role in binding or protection.

The importance of size and disorder in the cryoprotective effects of dehydrins.

Dehydrins protect plant proteins and membranes from damage during drought and cold. Vitis riparia K2 is a 48-residue protein that can protect lactate dehydrogenase from freeze-thaw damage by preventing the aggregation and denaturation of the enzyme. To further elucidate its mechanism, we used a series of V. riparia K2 concatemers (K4, K6, K8, and K10) and natural dehydrins (V. riparia YSK2, 60 kilodalton peach dehydrin [PCA60], barley dehydrin5 [Dhn5], Thellungiella salsuginea dehydrin2 [TsDHN-2], and Opuntia streptacantha dehydrin1 [OpsDHN-1]) to test the effect of the number of K-segments and dehydrin size on their ability to protect lactate dehydrogenase from freeze-thaw damage. The results show that the larger the hydrodynamic radius of the dehydrin, the more effective the cryoprotection. A similar trend is observed with polyethylene glycol, which would suggest that the protection is simply a nonspecific volume exclusion effect that can be manifested by any protein. However, structured proteins of a similar range of sizes did not show the same pattern and level of cryoprotection. Our results suggest that with respect to enzyme protection, dehydrins function primarily as molecular shields and that their intrinsic disorder is required for them to be an effective cryoprotectant. Lastly, we show that the cryoprotection by a dehydrin is not due to any antifreeze protein-like activity, as has been reported previously.

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.

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.

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.

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 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.

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 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.