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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Sequence and functional conservation of the intergenic region between the head-to-head genes encoding the small heat shock proteins alphaB-crystallin and HspB2 in the mammalian lineage.

An unexpected feature of the large mammalian genome is the frequent occurrence of closely linked head-to-head gene pairs. Close apposition of such gene pairs has been suggested to be due to sharing of regulatory elements. We show here that the head-to-head gene pair encoding two small heat shock proteins, alphaB-crystallin and HspB2, is closely linked in all major mammalian clades, suggesting that this close linkage is of selective advantage.

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.

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.

Influence of hormones and growth factors on lens protein composition: the effect of dexamethasone and PDGF-AA.

Purpose: To investigate the effect of hormones and ocular growth factors on the expression of alpha-, beta-, and gamma-crystallins in rat lens epithelial and fiber cells.

Methods: PDGF-AA, EGF, NGF, M-CSF, BMP-2, BMP-4, dexamethasone, and estrogen were tested for their ability to alter the spectrum of crystallins in explanted newborn rat lens epithelial cells or in vitro differentiating newborn rat lens fiber cells. The accumulation of alphaA-, aB-, betaA3/1-, betaB2-, and gamma-crystallin was measured by western blot and dot blot analysis.

Crystal structure of truncated human betaB1-crystallin.

Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.

Translational thermotolerance provided by small heat shock proteins is limited to cap-dependent initiation and inhibited by 2-aminopurine.

Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock.

Subunit exchange demonstrates a differential chaperone activity of calf alpha-crystallin toward beta LOW- and individual gamma-crystallins.

The chaperone activity of native alpha-crystallins toward beta(LOW)- and various gamma-crystallins at the onset of their denaturation, 60 and 66 degrees C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for one recombinant human gamma S. SAXS data demonstrated an irreversible doubling in molecular weight and a corresponding increase in size of alpha-crystallins at temperatures above 60 degrees C.