Nicolette H. Lubsen

The γ-crystallins and human cataracts : a puzzle made clearer

Despite the fact that cataracts constitute the leading cause of blindness worldwide, the mechanisms of lens opacification remain unclear. We recently mapped the aculeiform cataract to the γ-crystallin locus (CRYG) on chromosome 2q33-35, and mutational analysis of the CRYG-genes cluster identified the aculeiform-cataract mutation in exon 2 of γ-crystallin D (CRYGD). This mutation occurred in a highly conserved amino acid and could be associated with an impaired folding of CRYGD. During our study, we observed that the previously reported Coppock-like–cataract mutation, the first human cataract mutation, in the pseudogene CRYGE represented a polymorphism seen in 23% of our control population. Further analysis of the original Coppock-like–cataract family identified a missense mutation in a highly conserved segment of exon 2 of CRYGC. These mutations were not seen in a large control population. There is no direct evidence, to date, that up-regulation of a pseudogene causes cataracts. To our knowledge, these findings are the first evidence of an involvement of CRYGC and support the role of CRYGD in human cataract formation.

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.

Human alpha B-crystallin - Small heat shock protein and molecular chaperone

The polymerase chain reaction was used to amplify a cDNA sequence encoding the human αB-crystallin. The amplified cDNA fragment was cloned into the bacterial expression vector pMAL-c2 and expressed as a soluble fusion protein coupled to maltose-binding protein (MBP). After maltose affinity chromatography and cleavage from MBP by Factor Xa, the recombinant human αB-crystallin was separated from MBP and Factor Xa by anion exchange chromatography. Recombinant αB-crystallin was characterized by SDS-polyacrylamide electrophoresis (PAGE), Western immunoblot analysis, Edman degradation, circular dichroism spectroscopy, and size exclusion chromatography. The purified crystallin migrated on SDS-PAGE to an apparent molecular weight (Mr ∼22,000) that corresponded to total native human α-crystallin and was recognized on Western immunoblots by antiserum raised against human αB-crystallin purified from lens homogenates. Chemical sequencing, circular dichroism spectroscopy, and size exclusion chromatography demonstrated that the recombinant crystallin had properties similar or identical to its native counterpart. Both recombinant αB-crystallin and MBP-αB fusion protein associated to form high molecular weight complexes that displayed chaperone-like function by inhibiting the aggregation of alcohol dehydrogenase at 37°C and demonstrated the importance of the C-terminal domain of αB-crystallin for chaperone-like activity.

Human γ-crystallin genes: A gene family on its way to extinction☆

Abstract During hominoid evolution the γ-crystallins of the lens have decreased in quantity as well as complexity, a change correlated with an increased water content of the lens. To trace the molecular basis for the decrease in γ-crystallin gene expression, we have characterized the structure and expression of the human γ-crystallin gene family. We show that the human γ-crystallin gene family consists of six complete genes (γA, γB, γC, γD, ψγE and ψγF) and one second exon fragment, the γG gene. Model experiments showed that, although the γG sequence is bordered by consensus splice sites, it is most likely transcriptionally inactive in the lens. In the human embryonic lens the γC and γD genes accounted for 81 % of the γ-crystallin transcripts, the γA gene contributed 14% and the γB gene only 5%. The composition of the γ-crystallin mRNA pool changed only after birth, with the γD transcript as the only detectable transcript at ten years of age. The relative activities of the γA, γC and γD promoters in a transient expression system were in agreement with the ratio of their in vivo RNA levels, suggesting that the difference in accumulation of these transcripts is due to differences in the rate of transcription. The γB promoter was much more active than expected and had lost its tissue-specificity. Model experiments showed that the low yield of the γB transcript is due to post-transcriptional processes, most likely RNA instability mediated by third exon sequences. Together with previous data, our results show that the decrease in expression of the γ-crystallin genes in the human lens is the consequence of gene loss (γG), inactivation of coding sequences (ψγE and ψγF), decrease in rate of transcription (γA), increase in rate of RNA turn-over (γB) and a delay in the onset of transcription during development.

Concerted and divergent evolution within the rat γ-crystallin gene family☆

Abstract The nucleotide sequences of six rat γ-crystallin genes have been determined. All genes have the same mosaic structure: the first exons contain a relatively short (25 to 44 base-pair) 5′ non-coding region and the first nine base-pairs of the coding sequence, the second exons encode protein motifs I and II, while protein motifs III and IV are encoded by the third exons. The third exons also contain a 60 to 67-base-pair long 3′ non-coding region. In the γ1–2 gene, the splice acceptor site of the third exon has been shifted three base-pairs upstream. Hence, the protein product of this gene is one amino acid residue longer. The first introns, though varying in length from 85 to 100 base-pairs, are conserved in sequence. The second introns vary considerably in length (0.9 × 10 3 to 1.9 × 10 3 base-pairs) and sequence. The second exons of the genes show concerted evolution and have undergone multiple gene conversions. In contrast, the third exons show divergent evolution. From the sequences of the third exons, an evolutionary tree of the gene family was constructed. This tree suggests that three of the present genes derive directly from the genes that originated from a tandem duplication of a two-gene cluster. Two duplications of the last gene of the four-gene cluster then yielded the other three genes. Region a′ of the third exon, encoding protein motif III, is variable, while the region encoding protein motif IV (b′) is constant. We postulate that this variability in region a′ is due to a period of radiation after each gene duplication. A comparison of the rat sequences with those of orthologous sequences from other species shows that the variation in region a′ is now preserved. Hence, it might specify the specific functional property of each γ-crystallin protein within the lens.

Crystal structure of truncated human βB1-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 βγ‐crystallin family assemble into an array of oligomer sizes, forming intricate higher‐order networks in the lens cell. Here we describe the 1.4 Å resolution crystal structure of a truncated version of human βB1 that resembles an in vivo age‐related truncation. The structure shows that unlike its close homolog, βB2‐crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric γ‐crystallins. However, the four‐domain dimer resembles one half of the crystallographic bovine βB2 tetramer and is similar to the engineered circular permuted rat βB2. The crystal structure shows that the truncated βB1 dimer is extremely well suited to form higher‐order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions.

The stability of human acidic β-crystallin oligomers and hetero-oligomers

Abstract Crystallins are bulk structural proteins of the eye lens that have to last a life time. They gradually become modified with age, denature and form light scattering centres. High thermodynamic and kinetic stability of the crystallins enables them to resist unfolding and delay cataract. Here we have made recombinant human βA1-, βA3-, and βA4-crystallins. The βA3-crystallin formed higher oligomers that lead to precipitation at ambient temperature. Heat-induced precipitation of βA3-crystallin was compared with human and calf βB2-crystallins, showing that the human proteins start to precipitate above 50°C while the calf βB2-crystallin stays in solution even when unfolded. The stabilities of these human acidic β-crystallin homo-oligomers have been estimated by measuring their unfolding in urea at neutral pH. βA3/1/βB1 and βA4/βB1-crystallin hetero-oligomers have been prepared from homo-oligomers by subunit exchange. The resolution of the methodology used was insufficient to detect a stabilization of the βA4-crystallin subunit in the hetero-oligomer, the βA1-crystallin subunit was clearly stabilized by its interaction with βB1-crystallin. Circular dichroism and fluorescence spectroscopies show that homo-dimer surface tryptophans become buried in the βA3/1/βB1-crystallin hetero-dimer concomitant with changes in polypeptide chain conformation.

Developmental expression of crystallin genes: In situ hybridization reveals a differential localization of specific mRNAs☆

The time and place of the accumulation of alpha A-, beta B1- and gamma-crystallin RNA in the developing rat lens have been studied by in situ hybridization. alpha A- and gamma-crystallin RNA were first detected in the lens vesicle, while beta B1-crystallin RNA could be seen only after elongation of the primary fiber cells. Both beta B1- and gamma-crystallin RNA were confined to the fiber cells of fetal lenses, while alpha A-crystallin mRNA could also be detected in the epithelial cells. A quantification of the hybridization pattern obtained in the differentiation zone of the newborn rat lens showed that alpha A-crystallin RNA is concentrated in the cortical zone. alpha B-crystallin mRNA has the same distribution pattern. beta B1-crystallin RNA was relatively poorly detectable by in situ hybridization in both fetal and newborn rat lenses. The grain densities obtained with this probe increased from the periphery of the lens toward the interior, indicating that beta B1-crystallin RNA accumulated during differentiation of the secondary fiber cells. A similar accumulation pattern was obtained for gamma-crystallin mRNA, but, unexpectedly, this RNA could also be detected in the elongating epithelial cells. Our results show that gamma-crystallin RNA starts to accumulate as soon as visible elongation of epithelial cells occurs, during differentiation of the primary as well as the secondary fiber cells.

Rise and fall of crystallin gene messenger levels during fibroblast growth factor induced terminal differentiation of lens cells.

Explanted rat lens epithelial cells differentiate synchronously in vitro to lens fiber cells in the presence of basic fibroblast growth factor (bFGF). We have monitored the expression of the three rat crystallin gene families, the alpha-, beta-, and gamma-crystallin genes, during this process. The expression of these gene families is sequentially activated, first the alpha-crystallin genes at Day 1, then the beta-crystallin genes at Day 3, and finally the gamma-crystallin genes at Day 8. The steady state levels of alpha- and beta-crystallin mRNA are not affected by incubation with actinomycin D, suggesting that these mRNAs are stable. Nevertheless, all crystallin mRNAs disappear from the differentiated explants between Days 10 and 11, a process signaled by bFGF. At this time a novel abundant mRNA appears. Cloning and sequencing showed that this mRNA encoded aldose reductase. Our results suggest a novel model for the regulation of crystallin synthesis during lens cell differentiation: a gene pulse delivers a certain amount of stable mRNA, this mRNA is removed at a later stage of differentiation by a stage-specific breakdown mechanism. Each of these regulatory steps requires a signal from bFGF.