Knockdown of the small heat-shock protein p26 by RNA interference modifies the diapause proteome of Artemia franciscana.

Embryos of the crustacean Artemia franciscana may arrest as gastrulae, forming cysts that enter diapause, which is a state of reduced metabolism and enhanced stress tolerance. Diapausing cysts survive physiological stresses for years due, in part, to molecular chaperones. p26, a small heat-shock protein, is an abundant diapause-specific molecular chaperone in cysts, and it affects embryo development and stress tolerance. p26 is therefore thought to influence many proteins in cysts, and this study was undertaken to determine how the loss of p26 by RNA interference (RNAi) affects the diapause proteome of A. franciscana. The proteome was analyzed by shot-gun proteomics coupled to differential isotopic labeling and tandem mass spectrometry. Proteins in the diapause proteome included metabolic enzymes, antioxidants, binding proteins, structural proteins, transporters, translation factors, receptors, and signal transducers. Proteins within the diapause proteome either disappeared or were reduced in amount when p26 was knocked down, or conversely, proteins appeared or increased in amount. Those proteins that disappeared may be p26 substrates, whereas the synthesis of those proteins that appeared or increased may be regulated by p26. This study provides the first global characterization of the diapause proteome of A. franciscana and demonstrates that the sHsp p26 influences proteome composition.

ArHsp40 and ArHsp40-2 contribute to stress tolerance and longevity in Artemia franciscana, but only ArHsp40 influences diapause entry.

Embryos of the crustacean Artemia franciscana develop either ovoviviparously or oviparously, yielding swimming larvae (nauplii) or encysted gastrulae (cysts), respectively. Nauplii moult several times and become adults whereas cysts enter diapause, a state of dormancy characterized by exceptionally low metabolism and high stress tolerance. Synthesis of molecular chaperones such as the J-domain proteins ArHsp40 and ArHsp40-2 occurs during embryo development and post-diapause growth of A. franciscana and they influence development and stress tolerance. To further investigate J-domain protein function, ArHsp40 and ArHsp40-2 were each knocked down by RNA interference. Reductions in ArHsp40 and ArHsp40-2 had no effect on adult survival, time to release of cysts and nauplii from females and first-brood size. However, knockdown of both A. franciscana J-domain proteins reduced the longevity and heat tolerance of nauplii, with the loss of ArHsp40 having a greater effect. The knockdown of ArHsp40, but not of ArHsp40-2, caused approximately 50% of cysts to abort diapause entry and hatch without exposure to an exogenous signal such as low temperature and/or desiccation. Cysts lacking ArHsp40 that entered diapause exhibited decreased stress tolerance as did cysts with reduced ArHsp40-2, the latter to a lesser degree. The longevity of nauplii hatching prematurely from cysts was less than for nauplii arising by other means. The results expand our understanding of Hsp40 function in A. franciscana stress tolerance and development, especially during diapause, and they provide the first example of a molecular chaperone that influences diapause entry.

Insect heat shock proteins during stress and diapause.

Insect heat shock proteins include ATP-independent small heat shock proteins and the larger ATP-dependent proteins, Hsp70, Hsp90, and Hsp60. In concert with cochaperones and accessory proteins, heat shock proteins mediate essential activities such as protein folding, localization, and degradation. Heat shock proteins are synthesized constitutively in insects and induced by stressors such as heat, cold, crowding, and anoxia. Synthesis depends on the physiological state of the insect, but the common function of heat shock proteins, often working in networks, is to maintain cell homeostasis through interaction with substrate proteins. Stress-induced expression of heat shock protein genes occurs in a background of protein synthesis inhibition, but in the course of diapause, a state of dormancy and increased stress tolerance, these genes undergo differential regulation without the general disruption of protein production. During diapause, when ATP concentrations are low, heat shock proteins may sequester rather than fold proteins.

Artemin, a diapause-specific chaperone, contributes to the stress tolerance of Artemia franciscana cysts and influences their release from females.

Females of the crustacean Artemia franciscana produce either motile nauplii or gastrula stage embryos enclosed in a shell impermeable to nonvolatile compounds and known as cysts. The encysted embryos enter diapause, a state of greatly reduced metabolism and profound stress tolerance. Artemin, a diapause-specific ferritin homolog in cysts has molecular chaperone activity in vitro. Artemin represents 7.2% of soluble protein in cysts, approximately equal to the amount of p26, a small heat shock protein. However, there is almost twice as much artemin mRNA in cysts as compared with p26 mRNA, suggesting that artemin mRNA is translated less efficiently. RNA interference employing the injection of artemin double-stranded RNA into the egg sacs of A. franciscana females substantially reduced artemin mRNA and protein in cysts. Decreasing artemin diminished desiccation and freezing tolerance of cysts, demonstrating a role for this protein in stress resistance. Knockdown of artemin increased the time required for complete discharge of a brood of cysts carried within a female from a few hours up to 4 days, an effect weakened in successive broods. Artemin, an abundant molecular chaperone, contributes to stress tolerance of A. franciscana cysts while influencing their development and/or exit from females.

ArHsp90 is important in stress tolerance and embryo development of the brine shrimp, Artemia franciscana.

Females of the extremophile crustacean, Artemia franciscana, either release motile nauplii via the ovoviviparous pathway or encysted embryos (cysts) via the oviparous pathway. Cysts contain an abundant amount of the ATP-independent small heat shock protein that contributes to stress tolerance and embryo development, however, little is known of the role of ATP-dependent molecular chaperone, heat shock protein 90 (Hsp90) in the two processes. In this study, a hsp90 was cloned from A. franciscana. Characteristic domains of ArHsp90 were simulated from the deduced amino acid sequence, and 3D structures of ArHsp90 and Hsp90s of organisms from different groups were aligned. RNA interference was then employed to characterize ArHsp90 in A. franciscana nauplii and cysts. The partial knockdown of ArHsp90 slowed the development of nauplius-destined, but not cyst-destined embryos. ArHsp90 knockdown also reduced the survival and stress tolerance of nauplii newly released from A. franciscana females. Although the reduction of ArHsp90 had no effect on the development of diapause-destined embryos, the resulting cysts displayed reduced tolerance to desiccation and low temperature, two stresses normally encountered by A. franciscana in its natural environment. The results reveal that Hsp90 contributes to the development, growth, and stress tolerance of A. franciscana, an organism of practical importance as a feed source in aquaculture.

Priming the prophenoloxidase system of Artemia franciscana by heat shock proteins protects against Vibrio campbellii challenge.

Like other invertebrates, the brine shrimp Artemia franciscana relies solely on innate immunity, which by definition lacks adaptive characteristics, to combat against invading pathogens. One of the innate mechanisms is melanisation of bacteria mediated by the activation of the prophenoloxidase (proPO) system. The 70 kDa heat shock proteins (Hsp70) derived from either prokaryote (Escherichia coli) or eukaryote (Artemia), well conserved and immune-dominant molecules, protect Artemia against Vibrio campbellii. However, the molecular mechanisms by which these proteins protect Artemia against Vibrio campbellii infection are unknown. Here we demonstrated that feeding gnotobiotically grown Artemia with either Artemia Hsp70 or the E. coli Hsp70 equivalent DnaK, each overproduced in E. coli, followed by V. campbellii challenge enhanced the proPO system, at both mRNA and protein activity levels. Additionally, the Artemia fed with these proteins survived well in a Vibrio challenge assay. These results indicated that Hsp70s derived from either prokaryotic or eukaryotic sources generate protective immunity in the crustacean Artemia against V. campbellii infection by priming the proPO system. This is apparently the first in vivo report on priming activity of Hsp70 in an invertebrate.

Gene expression, metabolic regulation and stress tolerance during diapause.

Diapause entails molecular, physiological and morphological remodeling of living animals, culminating in a dormant state characterized by enhanced stress tolerance. Molecular mechanisms driving diapause resemble those responsible for biochemical processes in proliferating cells and include transcriptional, post-transcriptional and post-translational processes. The results are directed gene expression, differential mRNA and protein accumulation and protein modifications, including those that occur in response to changes in cellular redox potential. Biochemical pathways switch, metabolic products change and energy production is adjusted. Changes to biosynthetic activities result for example in the synthesis of molecular chaperones, late embryogenesis abundant (LEA) proteins and protective coverings, all contributing to stress tolerance. The purpose of this review is to consider regulatory and mechanistic strategies that are potentially key to metabolic control and stress tolerance during diapause, while remembering that organisms undergoing diapause are as diverse as the processes itself. Some of the parameters described have well-established roles in diapause, whereas the evidence for others is cursory.

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.

Improving the long-term storage of a mammalian biosensor cell line via genetic engineering.

The unique properties of mammalian cells make them valuable for a variety of applications in medicine, industry, and diagnostics. However, the utility of such cells is restricted due to the difficulty in storing them non-frozen for an extended time and still maintaining their stability and responsiveness. In order to extend the active life span of a mammalian biosensor cell line at room and refrigerated temperatures, we have over expressed genes that are reported to provide protection from apoptosis, stress, or oxidation. We demonstrated that over expression of genes from the extremophile, Artemia franciscana, as well as GADD45beta, extends room-temperature storage of fully active cells 3.5-fold, while over production of several anti-apoptotic proteins extended 4 degrees C storage 2- to 3-fold. Methodologies like these that improve the stability of mammalian-cell-based technologies in the absence of freezers may enable widespread use of these tools in applications that have been considered impractical based solely on limited storage characteristics.

Diapause termination and development of encysted Artemia embryos: roles for nitric oxide and hydrogen peroxide.

Encysted embryos (cysts) of the brine shrimp Artemia undergo diapause, a state of profound dormancy and enhanced stress tolerance. Upon exposure to the appropriate physical stimulus diapause terminates and embryos resume development. The regulation of diapause termination and post-diapause development is poorly understood at the molecular level, prompting this study on the capacity of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) to control these processes. Exposure to H(2)O(2) and NO, the latter generated by the use of three NO generators, promoted cyst development, emergence and hatching, effects nullified by catalase and the NO scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). The maximal effect of NO and H(2)O(2) on cyst development was achieved by 4 h of exposure to either chemical. NO was effective at a lower concentration than H(2)O(2) but more cysts developed in response to H(2)O(2). Promotion of development varied with incubation conditions, indicating for the first time a population of Artemia cysts potentially arrested in post-diapause and whose development was activated by either H(2)O(2) or NO. A second cyst sub-population, refractory to hatching after prolonged incubation, was considered to be in diapause, a condition broken by H(2)O(2) but not NO. These observations provide clues to the molecular mechanisms of diapause termination and development in Artemia, while enhancing the organism's value in aquaculture by affording a greater understanding of its growth and physiology.

Short-term cold stress and heat shock proteins in the crustacean Artemia franciscana.

In their role as molecular chaperones, heat shock proteins (Hsps) mediate protein folding thereby mitigating cellular damage caused by physiological and environmental stress. Nauplii of the crustacean Artemia franciscana respond to heat shock by producing Hsps; however, the effects of cold shock on Hsp levels in A. franciscana have not been investigated previously. The effect of cold shock at 1 °C followed by recovery at 27 °C on the amounts of ArHsp90, Hsp70, ArHsp40, and ArHsp40-2 mRNA and their respective proteins in A. franciscana nauplii was examined by quantitative PCR (qPCR) and immunoprobing of western blots. The same Hsp mRNAs and proteins were also quantified during incubation of nauplii at their optimal growth temperature of 27 °C. qPCR analyses indicated that the abundance of ArHsp90, Hsp70, and ArHsp40 mRNA remained relatively constant during both cold shock and recovery and was not significantly different compared with levels at optimal temperature. Western blotting revealed that ArHsp90, ArHsp40, and ArHsp40-2 were generally below baseline, but at detectable levels during the 6 h of cold shock, and persisted in early recovery stages before declining. Hsp70 was the only protein that remained constant in quantity throughout cold shock and recovery. By contrast, all Hsps declined rapidly during 6 h when nauplii were incubated continuously at 27 °C optimal temperature. Generally, the amounts of ArHsp90, ArHsp40, and ArHsp40-2 were higher during cold shock/recovery than those during continuous incubation at 27 °C. Our data support the conclusion that low temperature preserves Hsp levels, making them available to assist in protein repair and recovery after cold shock.

Cloning and sequencing of tubulin cDNAs from Artemia franciscana: evidence for differential expression of alpha- and beta-tubulin genes.

Tubulin is a heterodimeric protein composed of alpha- and beta-tubulin. In most organisms, they are encoded by multiple gene families whose members are subject to differential regulation. The objective of the work described herein was to better understand tubulin gene expression in the extremophile Artemia franciscana To this end tubulin cDNAs were cloned and sequenced. alphaAT2, an alpha-tubulin cDNA, differed by one nucleotide from alphaAT1, a previously cloned Artemia cDNA. This change, possibly generated by allelic variation, caused an M313V substitution in alpha-tubulin. The amino acid sequence of beta-tubulin encoded by betaAT1, one of only a very limited number of cloned crustacean beta-tubulin cDNA sequences yet available, and the first from Artemia, was similar to other beta-tubulins. However, betaAT1 possessed four degenerate TATA boxes in the 5' untranslated region, although authentic TATA and CCAAT boxes occurred in the 3' non-coding sequence. Analyses by quantitative PCR demonstrated that the amount of tubulin mRNA declined relative to total mRNA in progressive life history stages of Artemia and also that the organism contained more alphaAT2- than betaAT1-tubulin mRNA at all developmental phases examined.

ArHsp21, a developmentally regulated small heat-shock protein synthesized in diapausing embryos of Artemia franciscana.

Embryos of the crustacean, Artemia franciscana, undergo alternative developmental pathways, producing either larvae or encysted embryos (cysts). The cysts enter diapause, characterized by exceptionally high resistance to environmental stress, a condition thought to involve the sHSP (small heat-shock protein), p26. Subtractive hybridization has revealed another sHSP, termed ArHsp21, in diapause-destined Artemia embryos. ArHsp21 shares sequence similarity with p26 and sHSPs from other organisms, especially in the alpha-crystallin domain. ArHsp21 is the product of a single gene and its synthesis occurred exclusively in diapause-destined embryos. Specifically, ArHsp21 mRNA appeared 2 days post-fertilization, followed 1 day later by the protein, and then increased until embryo release at day 5. No ArHsp21 protein was detected in embryos developing directly into larvae, although there was a small amount of mRNA at 3 days post-fertilization. The protein was degraded during post-diapause development and had disappeared completely from second instar larvae. ArHsp21 formed large oligomers in encysted embryos and transformed bacteria. When purified from bacteria, ArHsp21 functioned as a molecular chaperone in vitro, preventing heat-induced aggregation of citrate synthase and reduction-driven denaturation of insulin. Sequence characteristics, synthesis patterns and functional properties demonstrate clearly that ArHsp21 is an sHSP able to chaperone other proteins and contribute to stress tolerance during diapause. As such, ArHsp21 would augment p26 chaperone activity and it may also possess novel activities that benefit Artemia embryos exposed to stress.

The synthesis of diapause-specific molecular chaperones in embryos of Artemia franciscana is determined by the quantity and location of heat shock factor 1 (Hsf1).

The crustacean, Artemia franciscana, displays a complex life history in which embryos either arrest development and undertake diapause as cysts or they develop into swimming nauplii. Diapause entry is preceded during embryogenesis by the synthesis of specific molecular chaperones, namely the small heat shock proteins p26, ArHsp21, and ArHsp22, and the ferritin homolog, artemin. Maximal synthesis of diapause-specific molecular chaperones is dependent on the transcription factor, heat shock factor 1 (Hsf1), found in similar amounts in cysts and nauplii newly released from females. This investigation was performed to determine why, if cysts and nauplii contain comparable amounts of Hsf1, only cyst-destined embryos synthesize diapause-specific molecular chaperones. Quantification by qPCR and immunoprobing of Western blots, respectively, demonstrated that hsf1 mRNA and Hsf1 peaked by day 2 post-fertilization in embryos that were developing into cysts and then declined. hsf1 mRNA and Hsf1 were present in nauplii-destined embryos on day 2 post-fertilization, but in much smaller amounts than in cyst-destined embryos, and they increased in quantity until release of nauplii from females. Immunofluorescent staining revealed that the amount of Hsf1 in nuclei was greatest on day 4 post-fertilization in cyst-destined embryos but could not be detected in nuclei of nauplius-destined embryos at this time. The differences in quantity and location of Hsf1 explain why embryos fated to become cysts and eventually enter diapause synthesize p26, ArHsp21, ArHsp22, and artemin, whereas nauplius-destined embryos do not produce these molecular chaperones.

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.

Gene expression in diapause-destined embryos of the crustacean, Artemia franciscana.

Diapause-destined embryos of the crustacean Artemia franciscana cease development as gastrulae, encyst, and enter a resting stage characterized by greatly reduced metabolic activity and extreme stress resistance. To better understand diapause induction and maintenance in Artemia embryos gene expression was analyzed by subtractive hybridization at two days post-fertilization, a time early in this developmental process. Eighty-five of 264 cDNA clones sequenced matched GenBank entries and they fell into categories designated as environmental information processing, cellular processes, genetic information processing and metabolism. Semi-quantitative RT-PCR of cDNAs populating the subtractive library identified seventeen up-regulated and four down-regulated transcripts, the former including those encoding a human transcription cofactor homologue, three small heat shock proteins, putative cell growth suppressor proteins and several enzymes. As examples, p8 may modulate gene expression during diapause in Artemia embryos. BRCA1 associated protein-1 (BAP1) and other functionally related proteins may influence cell growth and division during transition into diapause, a time when these processes are inhibited, whereas small heat shock proteins protect embryos from stress. This study represents the first systematic molecular characterization of diapause in crustaceans. Several differentially expressed genes were identified, expanding the repertoire of proteins potentially modified during diapause and suggesting mechanistic pathways indigenous to the initiation and maintenance of this physiological state.

Post-diapause synthesis of ArHsp40-2, a type 2 J-domain protein from Artemia franciscana, is developmentally regulated and induced by stress.

Post-diapause cysts of Artemia franciscana undergo a well-defined developmental process whereby internal differentiation leads to rupture of the cyst shell, release of membrane-enclosed nauplii and hatching to yield swimming larvae. The post-diapause development of A. franciscana has been examined at biochemical and molecular levels, yet little is known about molecular chaperone function during this process. In addressing this we recently described ArHsp40, a type 1 J-domain protein in post-diapause A. franciscana cysts and larvae. The current report describes ArHsp40-2, a second J-domain protein from A. franciscana. ArHsp40-2 is a type 2 J-domain protein, lacking a zinc binding domain but containing other domains characteristic of these proteins. Notably, ArHsp40-2 possesses a double barrel ß-domain structure in its substrate binding region, as does ArHsp40. qPCR revealed a relatively low amount of ArHsp40-2 mRNA in 0 h cysts which increased significantly until the E1 stage, most likely as a result of enhanced transcription, after which it declined. An antibody specific to ArHsp40-2 was produced and used to show that like its mRNA, ArHsp40-2 accumulated until the E1 stage and then decreased to amounts lower than those in 0 h cysts. The synthesis of ArHsp40-2 was induced by heat shock indicating that ArHsp40-2 is involved in stress resistance in cysts and nauplii. Accumulation in cysts during early post-diapause development followed by its sharp decline suggests a role in protein disaggregation/refolding, a function of Hsp40s from other organisms, where ArHsp40-2 assists in the rescue of proteins sequestered during diapause by p26, an abundant small heat shock protein (sHsp) in A. franciscana cysts.

Stress tolerance in diapausing embryos of Artemia franciscana is dependent on heat shock factor 1 (Hsf1).

Embryos of the crustacean, Artemia franciscana, may undergo oviparous development, forming encysted embryos (cysts) that are released from females and enter diapause, a state of suppressed metabolism and greatly enhanced stress tolerance. Diapause-destined embryos of A. franciscana synthesize three small heat shock proteins (sHsps), p26, ArHsp21 and ArHsp22, as well as artemin, a ferritin homologue, all lacking in embryos that develop directly into nauplii. Of these diapause-specific molecular chaperones, p26 and artemin are important contributors to the extraordinary stress tolerance of A. franciscana cysts, but how their synthesis is regulated is unknown. To address this issue, a cDNA for heat shock factor 1 (Hsf1), shown to encode a protein similar to Hsf1 from other organisms, was cloned from A. franciscana. Hsf1 was knocked down by RNA interference (RNAi) in nauplii and cysts of A. franciscana. Nauplii lacking Hsf1 died prematurely upon release from females, showing that this transcription factor is essential to the survival of nauplii. Diapause cysts with diminished amounts of Hsf1 were significantly less stress tolerant than cysts containing normal levels of Hsf1. Moreover, cysts deficient in Hsf1 possessed reduced amounts of p26, ArHsp21, ArHsp22 and artemin, revealing dependence on Hsf1 for expression of their genes and maximum stress tolerance. The results demonstrate an important role for Hsf1, likely in concert with other transcription factors, in the survival and growth of A. franciscana and in the developmentally regulated synthesis of proteins responsible for the stress tolerance of diapausing A. franciscana cysts.

Characterization of the microtubule proteome during post-diapause development of Artemia franciscana.

The microtubule proteome encompasses tubulin and a diverse group of proteins which associate with tubulin upon microtubule formation. These proteins either determine microtubule organization and function or their activity is influenced by microtubule association. To characterize the microtubule proteome in Artemia franciscana, tubulin assembly was induced with taxol in vitro after 0 and 12 h of post-diapause development. Proteins obtained by extraction of microtubules with 0.5 M NaCl were electrophoresed in two-dimensional gels and analyzed by mass spectrometry. Fifty-five proteins were identified with 10 of these occurring at both developmental stages, and multiple isoforms were observed for some proteins of the Artemia proteome. Their functions include roles in membrane transport, metabolism, chaperoning and protein synthesis, thus reflecting physiological properties of encysted Artemia such as stress resistance and the ability to rapidly initiate post-diapause development. For example, chaperones may protect tubulin during encystment and facilitate folding in metabolically active embryos. Additionally, the interaction of metabolic enzymes with microtubules funnels reaction intermediates, potentially enhancing efficiency within biochemical processes. This study represents the first systematic characterization of a crustacean microtubule proteome. Although it is difficult to be certain that all protein associations documented herein occur in vivo, the results suggest how protein-protein interactions contribute to cytoplasmic organization while implying how Artemia embryos resist stress and remain capable of development once diapause terminates.

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.