Activation of the antioxidant response in methionine deprived human cells results in an HSF1-independent increase in HSPA1A mRNA levels.

In cells starved for leucine, lysine or glutamine heat shock factor 1 (HSF1) is inactivated and the level of the transcripts of the HSF1 target genes HSPA1A (Hsp70) and DNAJB1 (Hsp40) drops. We show here that in HEK293 cells deprived of methionine HSF1 was similarly inactivated but that the level of HSPA1A and DNAJB1 mRNA increased. This increase was also seen in cells expressing a dominant negative HSF1 mutant (HSF379 or HSF1-K80Q), confirming that the increase is HSF1 independent. The antioxidant N-acetylcysteine completely inhibited the increase in HSPA1A and DNAJB1 mRNA levels upon methionine starvation, indicating that this increase is a response to oxidative stress resulting from a lack of methionine. Cells starved for methionine contained higher levels of c-Fos and FosB mRNA, but knockdown of these transcription factors had no effect on the HSPA1A or DNAJB1 mRNA level. Knockdown of NRF2 mRNA resulted in the inhibition of the increase in the HSPA1A mRNA, but not the DNAJB1 mRNA, level in methionine starved cells. We conclude that methionine deprivation results in both the amino acid deprivation response and an antioxidant response mediated at least in part by NRF2. This antioxidant response includes an HSF1 independent increase in the levels of HSPA1A and DNAJB1 mRNA.

A delayed antioxidant response in heat-stressed cells expressing a non-DNA binding HSF1 mutant.

To assess the consequences of inactivation of heat shock factor 1 (HSF1) during aging, we analyzed the effect of HSF1 K80Q, a mutant unable to bind DNA, and of dnHSF1, a mutant lacking the activation domain, on the transcriptome of cells 6 and 24 h after heat shock. The primary response to heat shock (6 h recovery), of which 30 % was HSF1-dependent, had decayed 24 h after heat shock in control cells but was extended in HSF1 K80Q and dnHSF1 cells. Comparison with literature data showed that even the HSF1 dependent primary stress response is largely cell specific. HSF1 K80Q, but not HSF1 siRNA-treated, cells showed a delayed stress response: an increase in transcript levels of HSF1 target genes 24 h after heat stress. Knockdown of NRF2, but not of ATF4, c-Fos or FosB, inhibited this delayed stress response. EEF1D_L siRNA inhibited both the delayed and the extended primary stress responses, but had off target effects. In control cells an antioxidant response (ARE binding, HMOX1 mRNA levels) was detected 6 h after heat shock; in HSF1 K80Q cells this response was delayed to 24 h and the ARE complex had a different mobility. Inactivation of HSF1 thus affects the timing and nature of the antioxidant response and NRF2 can activate at least some HSF1 target genes in the absence of HSF1 activity.

Heat shock factor 1 is inactivated by amino acid deprivation.

Mammalian cells respond to a lack of amino acids by activating a transcriptional program with the transcription factor ATF4 as one of the main actors. When cells are faced with cytoplasmic proteotoxic stress, a quite different transcriptional response is mounted, the heat shock response, which is mediated by HSF1. Here, we show that amino acid deprivation results in the inactivation of HSF1. In amino acid deprived cells, active HSF1 loses its DNA binding activity as demonstrated by EMSA and ChIP. A sharp decrease in the transcript level of HSF1 target genes such as HSPA1A (Hsp70), DNAJB1 (Hsp40), and HSP90AA1 is also seen. HSPA1A mRNA, but not DNAJB1 mRNA, was also destabilized. In cells cultured with limiting leucine, HSF1 activity also declined. Lack of amino acids thus could lead to a lower chaperoning capacity and cellular frailty. We show that the nutrient sensing response unit of the ASNS gene contains an HSF1 binding site, but we could not detect binding of HSF1 to this site in vivo. Expression of either an HSF1 mutant lacking the activation domain (HSF379) or an HSF1 mutant unable to bind DNA (K80Q) had only a minor effect on the transcript levels of amino acid deprivation responsive genes.

Protein refolding in peroxisomes is dependent upon an HSF1-regulated function.

Post-heat shock refolding of luciferase requires chaperones. Expression of a dominant negative HSF1 mutant (dnHSF1), which among other effects depletes cells of HSF1-regulated chaperones, blocked post-heat shock refolding of luciferase targeted to the cytoplasm, nucleus, or peroxisomes, while refolding of endoplasmic reticulum (ER)-targeted luciferase was inhibited by about 50 %. Luciferase refolding in the cytoplasm could be partially restored by expression of HSPA1A and fully by both HSPA1A and DNAJB1. For full refolding of ER luciferase, HSPA1A expression sufficed. Neither nuclear nor peroxisomal refolding was rescued by HSPA1A. A stimulatory effect of DNAJB1 on post-heat shock peroxisomal luciferase refolding was seen in control cells, while refolding in the cytoplasm or nucleus in control cells was inhibited by DNAJB1 expression in the absence of added HSPA1A. HSPB1 also improved refolding of peroxisomal luciferase in control cells, but not in dnHSF1 expressing cells. HSP90, HSPA5, HSPA6, and phosphomevalonate kinase (of which the synthesis is also downregulated by dnHSF1) had no effect on peroxisomal refolding in either control or chaperone-depleted cells. The chaperone requirement for post-heat shock refolding of peroxisomal luciferase in control cells is thus unusual in that it can be augmented by DNAJB1 or HSPB1 but not by HSPA1A; in dnHSF1 expressing cells, expression of none of the (co)-chaperones tested was effective, and an as yet to be identified, HSF1-regulated function is required.

sHSP in the eye lens: crystallin mutations, cataract and proteostasis.

α-Crystallin, a major component of the eye lens cytoplasm, is a large multimer formed from two members of the small heat shock protein (sHsp) family. Inherited crystallin mutations are a common cause of childhood cataract, whereas miscellaneous changes to the long-lived crystallins cause age-related cataract, the most common cause of blindness worldwide. Newly formed eye lens cells use proteostasis to deal with the consequences of mutations, whereas mature lens cells, devoid of the ATP-driven folding and degradation machines, are hypothesized to have the α-crystallin "holdase" chaperone function to prevent protein aggregation. We discuss the impact of truncating and missense mutations on α-crystallin, based on recent progress towards determining sHsp 3D structure. Dominant missense mutations to the "α-crystallin domain" of αA- (HSPB4) or αB-crystallin (HSPB5) occur on residues predicted to facilitate domain dynamics. αB-Crystallin is also expressed in striated muscle and mutations cause myopathy. The impact on these cellular cytoplasms is compared where sHsp multimer partners and metabolic constraints are different. Selected inherited mutations of the lens ß- and γ-crystallins are considered in the context of their possible dependence on the "holdase" chaperone function of α-crystallin. Looking at discrete changes to specific crystallin polypeptide chains that can function as chaperone or substrate provide insights into the workings of a cytoplasmic proteostatic system. These observations provide a framework for validating the function of α-crystallin as a chaperone, or as a lens space filler adapted from a chaperone function. Understanding the mechanistic role of α-crystallins will aid progress in research into age-related cataract and adult-onset myopathy. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

An atypical unfolded protein response in heat shocked cells.

BACKGROUND: The heat shock response (HSR) and the unfolded protein response (UPR) are both activated by proteotoxic stress, although in different compartments, and share cellular resources. How these resources are allocated when both responses are active is not known. Insight in possible crosstalk will help understanding the consequences of failure of these systems in (age-related) disease. RESULTS: In heat stressed HEK293 cells synthesis of the canonical UPR transcription factors XBP1s and ATF4 was detected as well as HSF1 independent activation of the promoters of the ER resident chaperones HSPA5 (BiP) and DNAJB9 (ERdj4). However, the heat stress activation of the DNAJB9 promoter, a XBP1s target, was not blocked in cells expressing a dominant negative IRE1α mutant, and thus did not require XBP1s. Furthermore, the DNA element required for heat stress activation of the DNAJB9 promoter is distinct from the ATF4 and ATF6 target elements; even though inhibition of eIF2α phosphorylation resulted in a decreased activation of the DNAJB9 promoter upon heat stress, suggesting a role for an eIF2α phosphorylation dependent product. CONCLUSIONS: The initial step in the UPR, synthesis of transcription factors, is activated by heat stress but the second step, transcriptional transactivation by these factors, is blocked and these pathways of the UPR are thus not productive. Expression of canonical ER chaperones is part of the response of heat stressed cells but another set of transcription factors has been recruited to regulate expression of these ER chaperones.

Explosive expansion of betagamma-crystallin genes in the ancestral vertebrate.

In jawed vertebrates, betagamma-crystallins are restricted to the eye lens and thus excellent markers of lens evolution. These betagamma-crystallins are four Greek key motifs/two domain proteins, whereas the urochordate betagamma-crystallin has a single domain. To trace the origin of the vertebrate betagamma-crystallin genes, we searched for homologues in the genomes of a jawless vertebrate (lamprey) and of a cephalochordate (lancelet). The lamprey genome contains orthologs of the gnathostome betaB1-, betaA2- and gammaN-crystallin genes and a single domain gammaN-crystallin-like gene. It contains at least two gamma-crystallin genes, but lacks the gnathostome gammaS-crystallin gene. The genome also encodes a non-lenticular protein containing betagamma-crystallin motifs, AIM1, also found in gnathostomes but not detectable in the uro- or cephalochordate genome. The four cephalochordate betagamma-crystallin genes found encode two-domain proteins. Unlike the vertebrate betagamma-crystallins but like the urochordate betagamma-crystallin, three of the predicted proteins contain calcium-binding sites. In the cephalochordate betagamma-crystallin genes, the introns are located within motif-encoding region, while in the urochordate and in the vertebrate betagamma-crystallin genes the introns are between motif- and/or domain encoding regions. Coincident with the evolution of the vertebrate lens an ancestral urochordate type betagamma-crystallin gene rapidly expanded and diverged in the ancestral vertebrate before the cyclostomes/gnathostomes split. The beta- and gammaN-crystallin genes were maintained in subsequent evolution, and, given the selection pressure imposed by accurate vision, must be essential for lens function. The gamma-crystallin genes show lineage specific expansion and contraction, presumably in adaptation to the demands on vision resulting from (changes in) lifestyle.

Co-chaperones are limiting in a depleted chaperone network.

To probe the limiting nodes in the chaperoning network which maintains cellular proteostasis, we expressed a dominant negative mutant of heat shock factor 1 (dnHSF1), the regulator of the cytoplasmic proteotoxic stress response. Microarray analysis of non-stressed dnHSF1 cells showed a two- or more fold decrease in the transcript level of 10 genes, amongst which are the (co-)chaperone genes HSP90AA1, HSPA6, DNAJB1 and HSPB1. Glucocorticoid signaling, which requires the Hsp70 and the Hsp90 folding machines, was severely impaired by dnHSF1, but fully rescued by expression of DNAJA1 or DNAJB1, and partially by ST13. Expression of DNAJB6, DNAJB8, HSPA1A, HSPB1, HSPB8, or STIP1 had no effect while HSP90AA1 even inhibited. PTGES3 (p23) inhibited only in control cells. Our results suggest that the DNAJ co-chaperones in particular become limiting in a depleted chaperoning network. Our results also suggest a difference between the transcriptomes of cells lacking HSF1 and cells expressing dnHSF1.

Manipulating heat shock factor-1 in Xenopus tadpoles: neuronal tissues are refractory to exogenous expression.

BACKGROUND: The aging related decline of heat shock factor-1 (HSF1) signaling may be causally related to protein aggregation diseases. To model such disease, we tried to cripple HSF1 signaling in the Xenopus tadpole. RESULTS: Over-expression of heat shock factor binding protein-1 did not inhibit the heat shock response in Xenopus. RNAi against HSF1 mRNA inhibited the heat shock response by 70% in Xenopus A6 cells, but failed in transgenic tadpoles. Expression of XHSF380, a dominant-negative HSF1 mutant, was embryonic lethal, which could be circumvented by delaying expression via a tetracycline inducible promoter. HSF1 signaling is thus essential for embryonic Xenopus development. Surprisingly, transgenic expression of the XHSF380 or of full length HSF1, whether driven by a ubiquitous or a neural specific promoter, was not detectable in the larval brain. CONCLUSIONS: Our finding that the majority of neurons, which have little endogenous HSF1, refused to accept transgene-driven expression of HSF1 or its mutant suggests that HSF1 levels are strictly controlled in neuronal tissue.

A DNAJB chaperone subfamily with HDAC-dependent activities suppresses toxic protein aggregation.

Misfolding and aggregation are associated with cytotoxicity in several protein folding diseases. A large network of molecular chaperones ensures protein quality control. Here, we show that within the Hsp70, Hsp110, and Hsp40 (DNAJ) chaperone families, members of a subclass of the DNAJB family (particularly DNAJB6b and DNAJB8) are superior suppressors of aggregation and toxicity of disease-associated polyglutamine proteins. The antiaggregation activity is largely independent of the N-terminal Hsp70-interacting J-domain. Rather, a C-terminal serine-rich (SSF-SST) region and the C-terminal tail are essential. The SSF-SST region is involved in substrate binding, formation of polydisperse oligomeric complexes, and interaction with histone deacetylases (HDAC4, HDAC6, SIRT2). Inhibiting HDAC4 reduced DNAJB8 function. DNAJB8 is (de)acetylated at two conserved C-terminal lysines that are not involved in substrate binding, but do play a role in suppressing protein aggregation. Combined, our data provide a functional link between HDACs and DNAJs in suppressing cytotoxic protein aggregation.

Hsp70 expression and induction as a readout for detection of immune modulatory components in food.

Stress proteins such as heat shock proteins (Hsps) are up-regulated in cells in response to various forms of stress, like thermal and oxidative stress and inflammation. Hsps prevent cellular damage and increase immunoregulation by the activation of anti-inflammatory T-cells. Decreased capacity for stress-induced Hsp expression is associated with immune disorders. Thus, therapeutic boosting Hsp expression might restore or enhance cellular stress resistance and immunoregulation. Especially food- or herb-derived phytonutrients may be attractive compounds to restore optimal Hsp expression in response to stress. In the present study, we explored three readout systems to monitor Hsp70 expression in a manner relevant for the immune system and evaluated novel Hsp co-inducers. First, intracellular staining and analysis by flow cytometry was used to detect stress and/or dietary compound induced Hsp70 expression in multiple rodent cell types efficiently. This system was used to screen a panel of food-derived extracts with potent anti-oxidant capacity. This strategy yielded the identity of several new enhancers of stress-induced Hsp70 expression, among them carvacrol, found in thyme and oregano. Second, CD4(+) T-cell hybridomas were generated that specifically recognized an immunodominant Hsp70 peptide. These hybridomas were used to show that carvacrol enhanced Hsp70 levels increased T-cell activation. Third, we generated a DNAJB1-luc-O23 reporter cell line to show that carvacrol increased the transcriptional activation of a heat shock promoter in the presence of arsenite. These assay systems are generally applicable to identify compounds that affect the Hsp level in cells of the immune system.

Differential neuroendocrine expression of multiple brain-derived neurotrophic factor transcripts.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin with important growth-promoting properties. We report here the first characterization of a BDNF gene in an amphibian, Xenopus laevis, and demonstrate that environmental factors can activate this gene in a promoter-specific fashion. The Xenopus BDNF gene contains six promoter-specific 5'-exons and one 3'-protein-encoding exon. We examined the expression of promoter-specific transcripts in Xenopus neuroendocrine melanotrope cells. These cells make a good model to study how environmental factors control gene expression. In animals placed on a black background melanotrope cells more actively produce and release alphaMSH than in animals on a white background. BDNF is cosequestered and coreleased with alphaMSH and stimulates biosynthesis of proopiomelanocortin (POMC), the precursor protein for alphaMSH. Our analysis of the expression of the BDNF transcripts revealed that there is differential use of some BDNF promoters in melanotrope cells, depending on the adaptation state of the frog. During black-background adaptation, stimulation of expression of BDNF transcript IV preceded that of the POMC transcript, suggesting the BDNF gene is an effector gene for POMC expression. The possible mechanisms regulating expression of the various transcripts are discussed on the basis of the potential calcium- and cAMP-responsive elements in the promoter region of exon IV. Finally, we show that the upstream open reading frames of BDNF transcripts I and IV markedly decrease BDNF translation efficiency, giving the first indication for a functional role of untranslated BDNF exons.

Protein folding includes oligomerization - examples from the endoplasmic reticulum and cytosol.

A correct three-dimensional structure is a prerequisite for protein functionality, and therefore for life. Thus, it is not surprising that our cells are packed with proteins that assist protein folding, the process in which the native three-dimensional structure is formed. In general, plasma membrane and secreted proteins, as well as those residing in compartments along the endocytic and exocytic pathways, fold and oligomerize in the endoplasmic reticulum. The proteins residing in the endoplasmic reticulum are specialized in the folding of this subset of proteins, which renders this compartment a protein-folding factory. This review focuses on protein folding in the endoplasmic reticulum, and discusses the challenge of oligomer formation in the endoplasmic reticulum as well as the cytosol.

Evolution of closely linked gene pairs in vertebrate genomes.

The orientation of closely linked genes in mammalian genomes is not random: there are more head-to-head (h2h) gene pairs than expected. To understand the origin of this enrichment in h2h gene pairs, we have analyzed the phylogenetic distribution of gene pairs separated by less than 600 bp of intergenic DNA (gene duos). We show here that a lack of head-to-tail (h2t) gene duos is an even more distinctive characteristic of mammalian genomes, with the platypus genome as the only exception. In nonmammalian vertebrate and in nonvertebrate genomes, the frequency of h2h, h2t, and tail-to-tail (t2t) gene duos is close to random. In tetrapod genomes, the h2t and t2t gene duos are more likely to be part of a larger gene cluster of closely spaced genes than h2h gene duos; in fish and urochordate genomes, the reverse is seen. In human and mouse tissues, the expression profiles of gene duos were skewed toward positive coexpression, irrespective of orientation. The organization of orthologs of both members of about 40% of the human gene duos could be traced in other species, enabling a prediction of the organization at the branch points of gnathostomes, tetrapods, amniotes, and euarchontoglires. The accumulation of h2h gene duos started in tetrapods, whereas that of h2t and t2t gene duos only started in amniotes. The apparent lack of evolutionary conservation of h2t and t2t gene duos relative to that of h2h gene duos is thus a result of their relatively late origin in the lineage leading to mammals; we show that once they are formed h2t and t2t gene duos are as stable as h2h gene duos.

mRNA made during heat shock enters the first round of translation.

To determine whether mRNA synthesized during a heat shock is translated at least once in spite of the strong inhibition of translation by heat shock, we used nonsense-mediated decay (NMD) as an assay since NMD requires a round of translation. As NMD substrate we used the human psigammaE-crystallin gene, which contains a premature termination codon, and as control, its close relative, the human gammaD-crystallin gene, both placed under control of the Hsp70 promoter. We show that no spliced psigammaE-crystallin mRNA can be detected in heat shocked cells, suggesting that NMD resumes as soon as splicing is restored. We further show that newly synthesized mRNAs co-sediment with the 40S ribosomal subunits, indicating that the transcripts are recruited to the translation machinery but are stalled at the translation initiation stage. Using fluorescence loss in photobleaching (FLIP) we show that cytoplasmic EGFP-CBP20 is immobile in heat shocked cells. CBP20 is part of the cap binding complex which is thought to direct the first round of translation. Together our data suggest that all mRNAs made during heat shock enter the pioneer round of translation.

In vivo heteromer formation. Expression of soluble betaA4-crystallin requires coexpression of a heteromeric partner.

The beta-crystallins are a family of long-lived, abundant structural proteins that are coexpressed in the vertebrate lens. As beta-crystallins form heteromers, a process that involves transient exposure of hydrophobic interfaces, we have examined whether in vivobeta-crystallin assembly is enhanced by protein chaperones, either small heat shock proteins, Hsp27 or alphaB-crystallin, or Hsp70. We show here that betaA4-crystallin is abundantly expressed in HeLa cells, but rapidly degraded, irrespective of the presence of Hsp27, alphaB-crystallin or Hsp70. Degradation is even enhanced by Hsp70. Coexpression of betaA4-crystallin with betaB2-crystallin yielded abundant soluble betaA4-betaB2-crystallin heteromers; betaB1-crystallin was much less effective in solubilizing betaA4-crystallin. As betaB2-crystallin competed for betaA4-crystallin with Hsp70 and the proteasomal degradation pathway, betaB2-crystallin probably captures an unstable betaA4-crystallin intermediate. We suggest that the proper folding of betaA4-crystallin is not mediated by general chaperones but requires a heteromeric partner, which then also acts as a dedicated chaperone towards betaA4-crystallin.

Hsp27 enhances recovery of splicing as well as rephosphorylation of SRp38 after heat shock.

A heat stress causes a rapid inhibition of splicing. Exogenous expression of Hsp27 did not prevent that inhibition but enhanced the recovery of splicing afterward. Another small heat shock protein, alphaB-crystallin, had no effect. Hsp27, but not alphaB-crystallin, also hastened rephosphorylation of SRp38-dephosphorylated a potent inhibitor of splicing-after a heat shock, although it did not prevent dephosphorylation by a heat shock. The effect of Hsp27 on rephosphorylation of SRp38 required phosphorylatable Hsp27. A Hsp90 client protein was required for the effect of Hsp27 on recovery of spicing and on rephosphorylation of SRp38. Raising the Hsp70 level by either a pre-heat shock or by exogenous expression had no effect on either dephosphorylation of SRp38 during heat shock or rephosphorylation after heat shock. The phosphatase inhibitor calyculin A prevented dephosphorylation of SRp38 during a heat shock and caused complete rephosphorylation of SRp38 after a heat shock, indicating that cells recovering from a heat shock are not deficient in kinase activity. Together our data show that the activity of Hsp27 in restoring splicing is not due to a general thermoprotective effect of Hsp27, but that Hsp27 is an active participant in the (de)phosphorylation cascade controlling the activity of the splicing regulator SRp38.

Urochordate betagamma-crystallin and the evolutionary origin of the vertebrate eye lens.

A refracting lens is a key component of our image-forming camera eye; however, its evolutionary origin is unknown because precursor structures appear absent in nonvertebrates. The vertebrate betagamma-crystallin genes encode abundant structural proteins critical for the function of the lens. We show that the urochordate Ciona intestinalis, which split from the vertebrate lineage before the evolution of the lens, has a single gene coding for a single domain monomeric betagamma-crystallin. The crystal structure of Ciona betagamma-crystallin is very similar to that of a vertebrate betagamma-crystallin domain, except for paired, occupied calcium binding sites. The Ciona betagamma-crystallin is only expressed in the palps and in the otolith, the pigmented sister cell of the light-sensing ocellus. The Ciona betagamma-crystallin promoter region targeted expression to the visual system, including lens, in transgenic Xenopus tadpoles. We conclude that the vertebrate betagamma-crystallins evolved from a single domain protein already expressed in the neuroectoderm of the prevertebrate ancestor. The conservation of the regulatory hierarchy controlling betagamma-crystallin expression between organisms with and without a lens shows that the evolutionary origin of the lens was based on co-option of pre-existing regulatory circuits controlling the expression of a key structural gene in a primitive light-sensing system.

Oligomerization and phase transitions in aqueous solutions of native and truncated human beta B1-crystallin.

Human betaB1-crystallin is a major eye-lens protein that undergoes in vivo truncation at the N-terminus with aging. By studying native betaB1 and truncated betaB1DeltaN41, which mimics an age-related in vivo truncation, we have determined quantitatively the effect of truncation on the oligomerization and phase transition properties of betaB1 aqueous solutions. The oligomerization studies show that the energy of attraction between the betaB1DeltaN41 proteins is about 10% greater than that of the betaB1 proteins. We have found that betaB1DeltaN41 aqueous solutions undergo two distinct types of phase transitions. The first phase transition involves an initial formation of thin rodlike assemblies, which then evolve to form crystals. The induction time for the formation of rodlike assemblies is sensitive to oligomerization. The second phase transition can be described as liquid-liquid phase separation (LLPS) accompanied by gelation within the protein-rich phase. We refer to this process as heterogeneous gelation. These two phase transitions are not observed in the case of betaB1 aqueous solutions. However, upon the addition of poly(ethylene glycol) (PEG), we observe heterogeneous gelation also for betaB1. Our PEG experiments allow us to estimate the difference in phase separation temperatures between betaB1 and betaB1DeltaN41. This difference is consistent with the increase in energy of attraction found in our oligomerization studies. Our work suggests that truncation is a cataractogenic modification since it favors protein condensation and the consequent formation of light scattering elements, and highlights the importance of the N-terminus of betaB1 in maintaining lens transparency.

Ageing and vision: structure, stability and function of lens crystallins.

The alpha-, beta- and gamma-crystallins are the major protein components of the vertebrate eye lens, alpha-crystallin as a molecular chaperone as well as a structural protein, beta- and gamma-crystallins as structural proteins. For the lens to be able to retain life-long transparency in the absence of protein turnover, the crystallins must meet not only the requirement of solubility associated with high cellular concentration but that of longevity as well. For proteins, longevity is commonly assumed to be correlated with long-term retention of native structure, which in turn can be due to inherent thermodynamic stability, efficient capture and refolding of non-native protein by chaperones, or a combination of both. Understanding how the specific interactions that confer intrinsic stability of the protein fold are combined with the stabilizing effect of protein assembly, and how the non-specific interactions and associations of the assemblies enable the generation of highly concentrated solutions, is thus of importance to understand the loss of transparency of the lens with age. Post-translational modification can have a major effect on protein stability but an emerging theme of the few studies of the effect of post-translational modification of the crystallins is one of solubility and assembly. Here we review the structure, assembly, interactions, stability and post-translational modifications of the crystallins, not only in isolation but also as part of a multi-component system. The available data are discussed in the context of the establishment, the maintenance and finally, with age, the loss of transparency of the lens. Understanding the structural basis of protein stability and interactions in the healthy eye lens is the route to solve the enormous medical and economical problem of cataract.