Ron P. H. Dirks

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.

Urochordate βγ-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 [1]. The vertebrate βγ-crystallin genes encode abundant structural proteins critical for the function of the lens [2]. 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 βγ-crystallin. The crystal structure of Ciona βγ-crystallin is very similar to that of a vertebrate βγ-crystallin domain, except for paired, occupied calcium binding sites. The Ciona βγ-crystallin is only expressed in the palps and in the otolith, the pigmented sister cell of the light-sensing ocellus. The Ciona βγ-crystallin promoter region targeted expression to the visual system, including lens, in transgenic Xenopus tadpoles. We conclude that the vertebrate βγ-crystallins evolved from a single domain protein already expressed in the neuroectoderm of the prevertebrate ancestor. The conservation of the regulatory hierarchy controlling βγ-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.

Cell-Autonomous TrkB Signaling in Presynaptic Retinal Ganglion Cells Mediates Axon Arbor Growth and Synapse Maturation during the Establishment of Retinotectal Synaptic Connectivity

BDNF contributes to the activity-dependent establishment and refinement of visual connectivity. In Xenopus, BDNF applications in the optic tectum influence retinal ganglion cell (RGC) axon branching and promote synapse formation and stabilization. The expression patterns of BDNF and TrkB suggest that BDNF specifically regulates the maturation of RGC axons at the target. It is possible, however, that BDNF modulates retinotectal synaptic connectivity by differentially influencing presynaptic RGC axons and postsynaptic tectal cells. Here, we combined single-cell expression of a dominant-negative TrkB–enhanced green fluorescent protein (GFP) fusion protein with confocal microscopy imaging in live Xenopus tadpoles to differentiate between presynaptic and postsynaptic actions of BDNF. Disruption of TrkB signaling in individual RGCs influenced the branching and synaptic maturation of presynaptic axon arbors. Specifically, GFP–TrkB.T1 overexpression increased the proportion of axons with immature, growth cone-like morphology, decreased axon branch stability, and increased axon arbor degeneration. In addition, GFP–TrkB.T1 overexpression reduced the number of red fluorescent protein–synaptobrevin-labeled presynaptic specializations per axon terminal. In contrast, overexpression of GFP–TrkB.T1 in tectal neurons did not alter synaptic number or the morphology or dynamic behavior of their dendritic arbors. Electron microscopy analysis revealed a significant decrease in the number of mature synaptic profiles and in the number of docked synaptic vesicles at retinotectal synapses made by RGC axons expressing GFP–TrkB.T1. Together, our results demonstrate that presynaptic TrkB signaling in RGCs is a key determinant in the establishment of visual connectivity and indicate that changes in tectal neuron synaptic connectivity are secondary to the BDNF-elicited enhanced stability and growth of presynaptic RGCs.

SIGNALS CONTROLLING THE EXPRESSION OF PDGF

PDGF is an important polypeptide growth factor that plays an essential role during early vertebrate development and is associated with tissue repair and wound healing in the adult vertebrate. Moreover, PDGF is thought to play a role in a variety of pathological phenomena, such as cancer, fibrosis and atherosclerosis. PDGF is expressed as a dimer of A and/or B chains, the precursors of which are encoded by two single copy genes. Although the PDGF genes are expressed coordinately in a number of cell types, they are independently expressed in a majority of cell types. The expression of either PDGF gene can be affected by very diverse extracellular stimuli and the type of response is dependent on the cell type that is exposed to the stimulus. Expression of the PDGF chains can be modulated at every imaginable level: by regulating accessibility of the transcription start site, by varying the transcription initiation rate, by using alternative transcription start sites, by alternative splicing, by using alternative polyadenylation signals, by varying mRNA decay rates, by regulating efficiency of translation, by protein modification, and by regulating secretion. Even upon secretion, the activity of PDGF can be modulated by non-specific or specific PDGF-binding proteins. This review provides an overview of the cell types in which the PDGF genes are expressed, of the factors that are known to affect the expression of PDGF, and of the various levels at which the expression of PDGF genes can be regulated.

Nucleotide sequence and expression of a β-tubulin gene from Plasmodium falciparum, a malarial parasite of man

Genomic and cDNA clones, containing a Plasmodium falciparum beta-tubulin coding sequence (pf-bTub), were isolated and characterized. Comparison of the genomic sequence with the cDNA sequence showed that the malarial bTub-coding region is interrupted by two introns, the positions of which are not found in any beta-tubulin gene (btub) from other species. The gene appears to be present as a single-copy gene in the P. falciparum genome and is expressed as a 2.3-kb transcript both in the asexual blood stages and in the sexual stages (gametes/zygotes) of the parasite. The deduced polypeptide product of the pf-btub gene is a protein of 445 amino acids (aa) (Mr 49,517). Comparison of the aa sequence of pf-bTub with that of bTubs from other species revealed that the malarial protein shows a high degree of similarity to mammalian bTubs. Upon examination of the colchicine-binding sites of pf-bTub we predict that this tubulin probably has an altered sensitivity to this inhibitor.

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.

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.

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.

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, αB-crystallin and HspB2, is closely linked in all major mammalian clades, suggesting that this close linkage is of selective advantage. Yet αB-crystallin is abundantly expressed in lens and muscle and in response to a heat shock, while HspB2 is abundant only in muscle and not upregulated by a heat shock. The intergenic distance between the genes for these two proteins in mammals ranges from 645xa0bp (platypus) to 1069xa0bp (opossum), with an average of about 900xa0bp; in chicken the distance was the same as in duck (1.6xa0kb). Phylogenetic footprinting and sequence alignment identified a number of conserved sequence elements close to the HspB2 promoter and two farther upstream. All known regulatory elements of the mouse αB-crystallin promoter are conserved, except in platypus and birds. The lens-specific region 1 (LSR1) and the heat shock elements (HSEs) lack in birds; in platypus the LSR1 is reduced to a Pax-6 site, while the Pax-6 site in LSR2 and a HSE are absent. Most likely the primordial mammalian αB-crystallin promoter had two LSRs and two HSEs. In transfection experiments the platypus αB-crystallin promoter retained heat shock responsiveness and lens expression. It also directed lens expression in Xenopus laevis transgenes, as did the HspB2 promoter of rat or blind mole rat. Deletion of the middle of the intergenic region including the upstream enhancer affected the activity of both the rat αB-crystallin and the HspB2 promoters, suggesting sharing of the enhancer region by the two promoters.

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.