Research into late embryogenesis abundant (LEA) proteins has been ongoing for more than 20 years but, although there is a strong association of LEA proteins with abiotic stress tolerance particularly dehydration and cold stress, for most of that time, their function has been entirely obscure. After their initial discovery in plant seeds, three major groups (numbered 1, 2 and 3) of LEA proteins have been described in a range of different plants and plant tissues. Homologues of groups 1 and 3 proteins have also been found in bacteria and in certain invertebrates. In this review, we present some new data, survey the biochemistry, biophysics and bioinformatics of the LEA proteins and highlight several possible functions. These include roles as antioxidants and as membrane and protein stabilisers during water stress, either by direct interaction or by acting as molecular shields. Along with other hydrophilic proteins and compatible solutes, LEA proteins might also serve as "space fillers" to prevent cellular collapse at low water activities. This multifunctional capacity of the LEA proteins is probably attributable in part to their structural plasticity, as they are largely lacking in secondary structure in the fully hydrated state, but can become more folded during water stress and/or through association with membrane surfaces. The challenge now facing researchers investigating these enigmatic proteins is to make sense of the various in vitro defined functions in the living cell: Are the LEA proteins truly multi-talented, or are they still just misunderstood?

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The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccationinduced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 -while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins -HSP70 can be co-immunoprecipitated with a polyglutaminecontaining client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability, but a bioinformatics survey does not support this, since there are no significant differences in the degree of disorder between desiccation tolerant and intolerant species. It seems

In spiders soluble proteins are converted to form insoluble silk fibres, stronger than steel. The final fibre product has long been the subject of study; however, little is known about the conversion process in the silk-producing gland of the spider. Here we describe a study of the conversion of the soluble form of the major spider-silk protein, spidroin, directly extracted from the silk gland, to a b-sheet enriched state using circular dichroism (CD) spectroscopy. Combined with electron microscopy (EM) data showing fibril formation in the b-sheet rich region of the gland and amino-acid sequence analyses linking spidroin and amyloids, these results lead us to suggest that the refolding conversion is amyloid like. We also propose that spider silk could be a valuable model system for testing hypotheses concerning b-sheet formation in other fibrilogenic systems, including amyloids.Keywords: spider silk; spidroin, amyloids; CD spectroscopy; low complexity peptides.Spiders have evolved sophisticated systems not only to produce and store proteins at high concentrations ( ‡ 30%) in solution, but also to control the conversion of these soluble proteins to form insoluble silk fibres [1]. The conversion process appears to rely on refolding of one or two members of the spidroin family of major silk proteins [2,3], but is poorly understood. Although the structure of the insect silkworm (Bombyx mori) silk has been well characterized [4] and a model [5] has been proposed for insect silk formation based on regenerated fibroin (the major protein in silkworm silk), the structure of spider silk is less well understood and appears to be different from insect silk in several respects [6]. Though progress has also been made in spinning fibres from recombinant spider silk produced in mammalian cells [7], the formation process in the spider has remained largely unexplored. Here, we describe the conversion of spidroin in the spider (Nephila edulis) using circular dichroism spectroscopy, electron microscopy and amino-acid sequence analyses. The results reveal a striking molecular-level similarity between spidersilk processing and amyloid-fibril formation. We conclude that the spider-silk production process, particularly the mechanisms that the spider employs to secrete, store and manipulate silk protein, could prove to be a valuable model system for exploring fibrilogenesis of amyloid, prion and other related proteins. E X P E R I M E N T A L P R O C E D U R E S Spider and sample preparationPrior to dissection, Nephila edulis female spiders were kept in plastic boxes (10 · 40 · 40 cm) and exclusively fed flies for several weeks. Under these conditions the spiders do not make webs thus ensuring a large accumulation of spidroin in their silk glands. The major-ampullate dragline-producing silk gland of a freshly decapitated spider was removed and the epithelium of the ampulla was carefully stripped off under spider Ringer solution (pH 7.4) [8]. The highly viscous remainder containing concentrated spidroin was separated into fractions deriv...

Glycogen is conventionally viewed as an energy reserve that can be rapidly mobilized for ATP production in higher organisms. However, several studies have noted that glycogen with short average chain length in some bacteria is degraded very slowly. In addition, slow utilization of glycogen is correlated with bacterial viability, that is, the slower the glycogen breakdown rate, the longer the bacterial survival time in the external environment under starvation conditions. We call that a durable energy storage mechanism (DESM). In this review, evidence from microbiology, biochemistry, and molecular biology will be assembled to support the hypothesis of glycogen as a durable energy storage compound. One method for testing the DESM hypothesis is proposed.

Background: AGR2 is a novel ER protein for which the molecular and cellular functions remain uncharacterized. Results: AGR2 associates to nascent chains in the ER, and its silencing impacts UPR and ERAD and sensitizes cells to autophagy. Conclusion: AGR2 plays an important role in the maintenance of ER homeostasis. Significance: AGR2-mediated control of ER homeostasis could be of importance for cancer development.

Sequences from ribosomal RNA (rRNA) genes have made a huge contribution to our current understanding of metazoan phylogeny and indeed the phylogeny of all of life. That said, some parts of this rRNA-based phylogeny remain unresolved. One approach to increase the resolution of these trees would be to use more appropriate models of sequence evolution in phylogenetic analysis. RNAs transcribed from rRNA genes have a complex secondary structure mediated by base pairing between sometimes distant regions of the rRNA molecule. The pairing between the stem nucleotides has important consequences for their evolution which differs from that of unpaired loop nucleotides. These differences in evolution should ideally be accounted for when using rRNA sequences for phylogeny estimation. We use a novel permutation approach to demonstrate the significant superiority of models of sequence evolution that allow stem and loop regions to evolve according to separate models and, in common with previous studies, we show that 16-state models that take base pairing of stems into account are significantly better than simpler, 4-state, single-nucleotide models. One of these 16-state models has been applied to the phylogeny of the Bilateria using small subunit rRNA (SSU) sequences. Our optimal tree largely echoes previous results based on SSU in particular supporting the tripartite Bilaterian tree of deuterostomes, lophotrochozoans, and ecdysozoans. There are also a number of differences, however, perhaps most important of which is the observation of a clade consisting of the gastrotrichs plus platyheminthes that is basal to all other lophotrochozoan taxa. Use of 16-state models also appears to reduce the Bayesian support given to certain biologically improbable groups found using standard 4-state models.

Multiple sequence alignment (MSA) is a crucial first step in the analysis of genomic and proteomic data. Commonly occurring sequence features, such as deletions and insertions, are known to affect the accuracy of MSA programs, but the extent to which alignment accuracy is affected by the positions of insertions and deletions has not been examined independently of other sources of sequence variation. We assessed the performance of 6 popular MSA programs (ClustalW, DIALIGN-T, MAFFT, MUSCLE, PROBCONS, and T-COFFEE) and one experimental program, PRANK, on amino acid sequences that differed only by short regions of deleted residues. The analysis showed that the absence of residues often led to an incorrect placement of gaps in the alignments, even though the sequences were otherwise identical. In data sets containing sequences with partially overlapping deletions, most MSA programs preferentially aligned the gaps vertically at the expense of incorrectly aligning residues in the flanking regions. Of the programs assessed, only DIALIGN-T was able to place overlapping gaps correctly relative to one another, but this was usually context dependent and was observed only in some of the data sets. In data sets containing sequences with non-overlapping deletions, both DIALIGN-T and MAFFT (G-INS-I) were able to align gaps with near-perfect accuracy, but only MAFFT produced the correct alignment consistently. The same was true for data sets that comprised isoforms of alternatively spliced gene products: both DIALIGN-T and MAFFT produced highly accurate alignments, with MAFFT being the more consistent of the 2 programs. Other programs, notably T-COFFEE and ClustalW, were less accurate. For all data sets, alignments produced by different MSA programs differed markedly, indicating that reliance on a single MSA program may give misleading results. It is therefore advisable to use more than one MSA program when dealing with sequences that may contain deletions or insertions, particularly for high-throughput and pipeline applications where manual refinement of each alignment is not practicable.