The Small Heat Shock Protein p26 Aids Development of Encysting Artemia Embryos, Prevents Spontaneous Diapause Termination and Protects against Stress

Artemia franciscana embryos enter diapause as encysted gastrulae, a physiological state of metabolic dormancy and enhanced stress resistance. The objective of this study was to use RNAi to investigate the function of p26, an abundant, diapause-specific small heat shock protein, in the development and behavior of encysted Artemia embryos (cysts). RNAi methodology was developed where injection of Artemia females with dsRNA specifically eliminated p26 from cysts. p26 mRNA and protein knock down were, respectively, confirmed by RT-PCR and immuno-probing of western blots. ArHsp21 and ArHsp22, diapause-related small heat shock proteins in Artemia cysts sharing a conserved α-crystallin domain with p26, were unaffected by injection of females with dsRNA for p26, demonstrating the specificity of protein knock down. Elimination of p26 delayed cyst release from females demonstrating that this molecular chaperone influences the development of diapause-destined embryos. Although development was slowed the metabolic activities of cysts either containing or lacking p26 were similar. p26 inhibited diapause termination after prolonged incubation of cysts in sea water perhaps by a direct effect on termination or indirectly because p26 is necessary for the preservation of diapause maintenance. Cyst diapause was however, terminated by desiccation and freezing, a procedure leading to high mortality within cyst populations lacking p26 and indicating the protein is required for stress tolerance. Cysts lacking p26 were also less resistant to heat shock. This is the first in vivo study to show that knock down of a small heat shock protein slows the development of diapause-destined embryos, suggesting a role for p26 in the developmental process. The same small heat shock protein prevents spontaneous termination of diapause and provides stress protection to encysted embryos.     

ArHsp22, a developmentally regulated small heat shock protein produced in diapause-destined Artemia embryos, is stress inducible in adults

Diapause embryos of the crustacean Artemia franciscana exhibit extreme stress tolerance, a property thought to involve molecular chaperones known as small heat shock proteins. To further explore this idea, the structure, function and synthesis of ArHsp22, an Artemia small heat shock protein, were characterized. ArHsp22 contains amino-terminal WXDPF motifs, an alpha-crystallin domain with a highly conserved arginine, and a carboxy-terminal I/VXI/V motif, all typical of small heat shock proteins. ArHsp22 formed large oligomers and exhibited molecular chaperone activity in vitro, protecting citrate synthase and insulin from denaturation. Quantitative PCR and immunoprobing of western blots revealed that ArHsp22 synthesis is restricted to diapause-destined Artemia embryos and that the protein is degraded during post-diapause development. ArHsp22 was observed in cyst nuclei, a location shared by p26 but not ArHsp21, which are two other diapause-specific Artemia small heat shock proteins. ArHsp22 production was enhanced by thermal stress, but only in adults, thus representing the first crustacean small heat shock protein whose synthesis is known to be both developmentally regulated and stress inducible. The results demonstrate that expression of the gene for ArHsp22 is modulated by multiple cues that vary with life history stage. Such findings are of importance in understanding diapause maintenance in Artemia embryos and the survival of adult animals experiencing environmental insult.     

Ontogeny of low molecular weight stress protein p26 during early development of the brine shrimp, Artemia franciscana

Embryogenesis in the brine shrimp, Artemia sp., occurs by one of two pathways: (i) the direct, uninterrupted development of nauplius larvae within the female or (ii) the production of embryos that arrest development at the gastrula stage and enter diapause. Diapause embryos are released from females into the aqueous environment where they remain in diapause until activated by appropriate environmental cues and resume development. These encysted embryos possess at least one low molecular weight stress protein, which we refer to as p26 and which has been implicated previously in the stress response of activated embryos. We investigated the appearance of p26 in developing diapause embryos in utero and looked for its presence in embryos developing directly into nauplii. We found p26 to be specific to diapause-destined embryos; it was not detected in direct-developing embryos. We conclude that p26 is not required for the basic developmental program that produces the nauplius. In diapause-destined embryos, p26 was first detectable after 3 days of development, at which time the embryos were late gastrulae. This protein continues to increase in amount until the encysted embryos are released, approximately 5 days after fertilization. At the time of release almost all p26 is located in the low speed supernatant fraction, but as released embryos continue diapause, p26 transfers to the pelleted nuclear fraction in increasing amounts. Our working hypothesis views p26 as a molecular chaperone preventing protein denaturation and aggregation under conditions associated with metabolic arrest and other stressful states, which these encysted embryos encounter.     

Molecular chaperones, stress resistance and development in Artemia franciscana

Embryos of the brine shrimp, Artemia franciscana, either develop directly into swimming larvae or are released from females as encysted gastrulae (cysts) which enter diapause, a reversible state of dormancy. Metabolic activity in diapause cysts is very low and these embryos are remarkably resistant to physiological stresses. Encysting embryos, but not those undergoing uninterrupted development, synthesize large amounts of two proteins, namely p26 and artemin. Cloning and sequencing demonstrated p26 is a small heat shock/alpha-crystallin protein while artemin has structural similarity to ferritin. p26 exhibits molecular chaperone activity in vitro, moves reversibly into nuclei during stress and confers thermotolerance on transformed organisms, suggesting critical roles in cyst development. The function of artemin is unknown. Encysted Artemia also contain an abundance of trehalose, a disaccharide capable of protecting embryos. Artemia represent a novel experimental system where the developmental functions of small heat shock/alpha-crystallin proteins and other stress response elements can be explored.     

Functional analysis of a small heat shock/alpha-crystallin protein from Artemia franciscana. Oligomerization and thermotolerance.

Oviparously developing embryos of the brine shrimp, Artemia franciscana, synthesize abundant quantities of a small heat shock/alpha-crystallin protein, termed p26. Wild-type p26 functions as a molecular chaperone in vitro and is thought to help encysted Artemia embryos survive severe physiological stress encountered during diapause and anoxia. Full-length and truncated p26 cDNA derivatives were generated by PCR amplification of p26-3-6-3, then cloned in either pET21(+) or pRSETC and expressed in Escherichia coli BL21(DE3). All constructs gave a polypeptide detectable on Western blots with either p26 specific antibody, or with antibody to the His(6) epitope tag encoded by pRSETC. Full-length p26 in cell-free extracts of E. coli was about equal in mass to that found in Artemia embryos, but p26 lacking N- and C-terminal residues remained either as monomers or small multimers. All p26 constructs conferred thermotolerance on transformed E. coli, although not all formed oligomers, and cells expressing N-terminal truncated derivatives of p26 were more heat resistant than bacteria expressing p26 with C-terminal deletions. The C-terminal extension of p26 is seemingly more important for thermotolerance than is the N-terminus, and p26 protects E. coli against heat shock when oligomer size and protein concentration are low. The findings have important implications for understanding the functional mechanisms of small heat shock/alpha-crystallin proteins.     

Stress-related proteins compared in diapause and in activated, anoxic encysted embryos of the animal extremophile, Artemia franciscana

Previous work indicated similarities between diapause and anoxic quiescence in encysted embryos (cysts) of the brine shrimp Artemia franciscana. That possibility was examined further in the present study through an immunochemical study of the following stress-related proteins in low speed supernatants and pellets: hsc70, artemin, p26, hsp21, LEA Group 1 protein and p8. Changes in the amounts and locations of these proteins occurred during the initial period after release of diapause cysts from females, and after activated (diapause-terminated) cysts were made anoxic. However, with the passage of incubation time the patterns seen in both kinds of cysts were more similar than different, lending further support to the possibility that activated anoxic embryos retain many of the mechanisms operative in the previous diapause condition.     

Diversity, structure, and expression of the gene for p26, a small heat shock protein from Artemia

p26, a small heat shock protein, is thought to protect Artemia embryos from stress during encystment and diapause. Full-length p26 cDNAs were compared and used to determine phylogenetic relationships between several Artemia species. The alpha-crystallin domain of p26 was the most conserved region of the protein and p26 from each Artemia species contained characteristic amino-terminal WD/EPF and carboxy-terminal VPI motifs. Sequence conservation suggested the importance of p26 to oviparously developing Artemia embryos and indicated common functions for the protein during development and stress resistance, although as shown by modeling some species-specific p26 amino acid substitutions may have adaptive significance. The p26 gene obtained from A. franciscana exhibited a unique sHSP intron arrangement with an intron in the 5'-untranslated region. Computer-assisted analysis revealed heat shock elements and other putative cis regulatory sequences but their role in gene regulation is unknown. In contrast to previous results for which Northern blots were analyzed, p26 gene expression was observed in ovoviviparous embryos by use of PCR-based methodology, but the p26 protein was not detected.     

Structural and functional roles for beta-strand 7 in the alpha-crystallin domain of p26, a polydisperse small heat shock protein from Artemia franciscana

Oviparous development in the extremophile crustacean, Artemia franciscana, generates encysted embryos which enter a profound state of dormancy, termed diapause. Encystment is marked by the synthesis of p26, a polydisperse small heat shock protein thought to protect embryos from stress. In order to elucidate structural/functional relationships within p26 and other polydisperse small heat shock proteins, and to better define the protein's role during diapause, amino acid substitutions R110G, F112R, R114A and Y116D were generated within the p26 alpha-crystallin domain by site-directed mutagenesis. These residues were chosen because they are highly conserved across species boundaries, and molecular modelling indicates that they are part of a key structural interface between dimers. The F112R mutation, which had the greatest impact on oligomerization, placed two charged residues at the p26 dimer-dimer interface, demonstrating the importance of beta-strand 7 in tetramer formation. All mutated versions of p26 were less able than wild-type p26 to confer thermotolerance on transformed bacteria and they exhibited diminished chaperone action in three in vitro assays; however, all variants retained protective activity. This apparent stability of p26 may, by prolonging effective chaperone life in vivo, enhance embryo stress resistance. All substitutions modified p26 intrinsic fluorescence, surface hydrophobicity and secondary structure, and the pronounced changes in variant R114A, as indicated by these physical measurements, correlated with the greatest loss of function. Although mutation R114A had the greatest effect on p26 chaperoning, it had the least on oligomerization. These results demonstrate that in contrast to many other small heat shock proteins, p26 effectiveness as a chaperone is independent of oligomerization. The results also reinforce the idea, occasioned by modelling, that R114 is removed slightly from dimer-dimer interfaces. Moreover, beta-strand 7 is shown to have an important role in oligomerization of p26, a function first proposed for this structural element upon crystallization of wheat Hsp16.9, a small heat shock protein with different quaternary structure.     

ArHsp40, a type 1 J-domain protein<Emphasis Type="Italic">,</Emphasis> is developmentally regulated and stress inducible in post-diapause <Emphasis Type="Italic">Artemia franciscana</Emphasis>

Upon diapause termination and exposure to favorable environmental conditions, cysts of the crustacean Artemia franciscana reinitiate development, a process dependent on the resumption of metabolic activity and the maintenance of protein homeostasis. The objective of the work described herein was to characterize molecular chaperones during post-diapause growth of A. franciscana. An Hsp40 complementary DNA (cDNA) termed ArHsp40 was cloned and shown to encode a protein with an amino-terminal J-domain containing a conserved histidine, proline, and aspartic acid (HPD) motif. Following the J-domain was a Gly/Phe (G/F) rich domain, a zinc-binding domain which contained a modified CXXCXGXG motif, and the carboxyl-terminal substrate binding region, all characteristics of type I Hsp40. Multiple alignment and protein modeling showed that ArHsp40 is comparable to Hsp40s from other eukaryotes and likely to be functionally similar. qRT-PCR revealed that during post-diapause development, ArHsp40 messenger RNA (mRNA) varied slightly until the E2/E3 stage and decreased significantly upon hatching. The immunoprobing of Western blots demonstrated that ArHsp40 was also relatively constant until E2/E3 and then declined dramatically. The drop in ArHsp40 when metabolism and protein synthesis were increasing was unexpected and demonstrated developmental regulation. The reduction in ArHsp40 at such an active life history stage indicates, as one possibility, that A. franciscana possesses additional Hsp40s, one or more of which replaces ArHsp40 as development progresses. Increased synthesis upon heat shock established that in addition to being developmentally regulated, ArHsp40 is stress inducible and, because it is found in mature cysts, ArHsp40 has the potential to contribute to stress tolerance during diapause.     

A small heat shock/alpha-crystallin protein from encysted Artemia embryos suppresses tubulin denaturation

Small heat shock/alpha-crystallin proteins function as molecular chaperones, protecting other proteins from irreversible denaturation by an energy-independent process. The brine shrimp, Artemia franciscana, produces a small heat shock/alpha-crystallin protein termed p26, found in embryos undergoing encystment, diapause, and metabolic arrest. These embryos withstand long-term anoxia and other stresses normally expected to cause death, a property likely dependent on molecular chaperone activity. The association of p26 with tubulin in unfractionated cell-free extracts of Artemia embryos was established by affinity chromatography, suggesting that p26 chaperones tubulin during encystment. To test this possibility, both proteins were purified by modifying published protocols, thereby simplifying the procedures, enhancing p26 yield about 2-fold, and recovering less tubulin than before. The denaturation of purified tubulin as it "aged" and exposed hydrophobic sites during incubation at 35 degrees C was greatly reduced when p26 was present; however, tubulin polymerization into microtubules was reduced. On incubation at 35 degrees C, centrifugation in sucrose density gradients demonstrated the association of purified p26 with tubulin. This is the first study where the relationship between a small heat shock/alpha-crystallin protein and tubulin from the same physiologically stressed organism was examined. The results support the proposal that p26 binds tubulin and prevents its denaturation, thereby increasing the resistance of encysted Artemia embryos to stress. Additional factors are apparently required for release of tubulin from p26 and restoration of efficient assembly, events that would occur as embryos resume development and the need for microtubules is established.     

Developmentally regulated gene expression in Artemia: Histone gene expression in newly hatched larvae

The synthesis of histones and presence of histone mRNA sequences in embryos and larvae of the brine shrimp, Artemia, were investigated. Radiolabeling of proteins synthesized in vivo followed by electrophoretic and fluorographic analysis confirmed the prediction that histone synthesis is coordinated with the wave of DNA replication in newly hatched larvae. No histone synthesis occurs during development of encysted embryos. Hybridization of cloned Artemia histone gene DNA to total cell RNA indicated that dormant encysted embryos do not contain “masked” messenger RNA.     

Comparisons of stress proteins and soluble carbohydrate in encysted embryos of Artemia franciscana and two species of Parartemia

We compared stress proteins (p26, artemin, hsp70) and alcohol-soluble carbohydrates (ASC) in cysts of Artemia franciscana and two as yet un-named species populations of Parartemia, the brine shrimp endemic to Australia. The small stress proteins and molecular chaperones, p26 and artemin, previously thought to be restricted to Artemia, and present in very large amounts in its encysted embryos (cysts), were also detected by western blotting in Parartemia cysts, even though roughly 85-100 million years have passed since these genera diverged. We interpret this finding as further evidence for the adaptive importance of these proteins in coping with the severe stresses these encysted embryos endure. As expected, hsp70 was present in all three groups of cysts, but apparently at somewhat lower concentrations in those of Parartemia. Based on measurements of ASC we propose that the disaccharide trehalose, critical for desiccation tolerance in many animal cells, has probably also been maintained in the metabolic repertoire of Parartemia whose cysts have well developed tolerance to severe desiccation.     

Oligomerization, chaperone activity, and nuclear localization of p26, a small heat shock protein from Artemia franciscana

Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 alpha-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers. The amino terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning. Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.     

Deubiquitinating enzyme BAP1 is involved in the formation and maintenance of the diapause embryos of Artemia

The modification of proteins by ubiquitination and deubiquitination plays an important role in various cellular processes. BRCA1-associated protein-1 (BAP1) is a deubiquitinating enzyme whose function in the control of the cell cycle requires both its deubiquitinating activity and nuclear localization. In the present study, a ubiquitin carboxyl-terminal hydrolase belonging to the BAP1 family was identified and characterized from Artemia parthenogenetica, a member of a family of brine shrimp that, under certain conditions, produce and release diapause embryos in which cell division and turnover of macromolecules are arrested. Western blot analysis and in vitro enzyme activity assay revealed ArBAP1 to be a cytoplasmic protein with typical ubiquitin hydrolase activity. Northern blot analysis revealed that ArBAP1 was abundant in the abdomen of Artemia producing diapause-destined embryos. Furthermore, by in situ hybridization, ArBAP1 was located exclusively in the embryos. In vivo knockdown of ArBAP1 by RNA interference resulted in the formation of embryos with split shells and abortive nauplii. The present findings suggest that ArBAP1, the first reported cytoplasmic BAP1, participates in the formation of diapause embryos and plays an important role in the control of cell cycle arrest in these encysted embryos.     

Functional characterization of artemin, a ferritin homolog synthesized in Artemia embryos during encystment and diapause

Oviparously developing embryos of the crustacean Artemia franciscana encyst and enter diapause, exhibiting a level of stress tolerance seldom seen in metazoans. The extraordinary stress resistance of encysted Artemia embryos is thought to depend in part on the regulated synthesis of artemin, a ferritin superfamily member. The objective of this study was to better understand artemin function, and to this end the protein was synthesized in Escherichia coli and purified to apparent homogeneity. Purified artemin consisted of oligomers approximately 700 kDa in molecular mass that dissociated into monomers and a small number of dimers upon SDS/PAGE. Artemin inhibited heat-induced aggregation of citrate synthase in vitro, an activity characteristic of molecular chaperones and shown here to be shared by apoferritin and ferritin. This is the first report that apoferritin/ferritin may protect cells from stress other than by iron sequestration. Stably transfected mammalian cells synthesizing artemin were more resistant to heat and H(2)O(2) than were cells transfected with vector only, actions also shared by molecular chaperones such as the small heat shock proteins. The data indicate that artemin is a structurally modified ferritin arising either from a common ancestor gene or by duplication of the ferritin gene. Divergence, including acquisition of a C-terminal peptide extension and ferroxidase center modification, eliminated iron sequestration, but chaperone activity was retained. Therefore, because artemin accumulates abundantly during development, it has the potential to protect embryos from stress during encystment and diapause without adversely affecting iron metabolism.     

Truncation attenuates molecular chaperoning and apoptosis inhibition by p26, a small heat shock protein from Artemia franciscana

The small heat shock proteins (sHSPs), which prevent irreversible protein denaturation and inhibit apoptosis, consist of an amino-terminus, the canonical α-crystallin domain, and a carboxy-terminal extension. It remains difficult, however, to define sHSP structure-function relationships and with this in mind p26, an sHSP from the crustacean Artemia franciscana, was truncated by deletion mutagenesis. Wild-type p26 cDNA and three truncated variants inserted into the eukaryotic expression vector pcDNA3.1/HisC were used to generate stably transfected 293H cells. p26 shielded transfected cells against death upon exposure to heat and oxidative stress. Truncation reduced chaperone activity, with cells synthesizing the p26 α-crystallin domain being the least resistant. Wild-type p26 inhibited apoptosis in transfected cells, with protection against oxidation-generated apoptosis being more effective than that against heat-induced apoptosis. Truncation reduced p26 apoptotic inhibitory activity, with the α-crystallin domain again being the least effective. The results show that a crustacean sHSP functions effectively in mammalian cells, demonstrating interchangeability of these proteins between distantly related organisms and indicating similarities in their mechanisms of action. Moreover, maximal activity was observed for full-length p26, indicating that structural elements required for chaperone activity and apoptosis inhibition reside throughout the protein.     

Influence of temperature and darkness on embryonic diapause termination in dormant Artemia cysts that have never been desiccated

Environmental cues for embryonic diapause termination (EDT) were investigated in the laboratory-produced encysted dormant embryos of the brine shrimp, Artemia franciscana. The cysts were spawned and kept throughout in a 2% sea salt solution. They were activated by various temperatures of the temperate zones or by continuous dark condition (DD), resulting in a state of EDT, and were thereafter able to resume their subsequent development and hatch under appropriate conditions. The level of EDT was conveniently assayed by a hatch test observed within 2 days at 28 degrees C under continuous light condition (LL). A cold treatment of the newly spawned dormant cysts, at 4 degrees C under DD for more than 100 days, resulted in more than 95% hatch of the dormant cysts. Similar treatments of the dormant cysts but at room temperature or 28 degrees C led to significantly different results (30-40% hatch). Almost all the residual non-hatched cysts derived from the above could hatch after an additional cold treatment (at 4 degrees C under DD for about 100 days). This might prove to be latent partial bivoltine in Artemia. Meanwhile, a rearing condition (28 degrees C under LL) induced the newly spawned cysts to hatch scatteredly at and after 1 month, resulting in 22% cumulative hatch on the 92nd day after spawning. When the newly spawned dormant cysts were pretreated at 28 degrees C under DD for 5 or 14 days and then reared at 28 degrees C under LL, the cumulative hatch significantly increased (60%). These results are discussed with respect to probable diapause regulator(s) involved in EDT.