Roy A. Quinlan

Chaperone activity of alpha-crystallins modulates intermediate filament assembly.

Intermediate filaments are generally regarded as one of the most insoluble and resilient cytoskeletal structures of eukaryotic cells. In extracts from the ocular lens, we noticed an unusually high level of vimentin in a soluble, non‐filamentous form. Immunoprecipitation of this soluble vimentin resulted in the co‐precipitation of alpha‐crystallins. The alpha‐crystallins are homologous to the small heat shock proteins (sHSPs) and have recently been identified as molecular chaperones, capable of preventing the heat‐induced aggregation of proteins. We find that the alpha‐crystallins dramatically inhibit the in vitro assembly of GFAP and vimentin in an ATP‐independent manner. This inhibition is also independent of the phosphorylation state of the alpha‐crystallin polypeptides and each one of the four polypeptides, either alpha A1‐, alpha A2‐, alpha B1‐ or alpha B2‐crystallin, are equally effective in this inhibition. Furthermore, we show that alpha‐crystallins can increase the soluble pool of GFAP when added to preformed filaments. Electron microscopy demonstrated that alpha‐crystallin particles could bind to intermediate filaments in a regular fashion, the spacing coinciding with the molecular length of GFAP. This is the first report, as far as we are aware, of a chaperone being involved in intermediate filament assembly and implicates chaperones in the remodeling of intermediate filaments during development and cell differentiation.

Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans.

Congenital cataracts are an important cause of bilateral visual impairment in infants. In a four-generation family of English descent, we mapped dominant congenital posterior polar cataract to chromosome 11q22-q22.3. The maximum LOD score, 3.92 at recombination fraction 0, was obtained for marker D11S898, near the gene that encodes crystallin alpha-B protein (CRYAB). By sequencing the coding regions of CRYAB, we found in exon 3 a deletion mutation, 450delA, that is associated with cataract in this family. The mutation resulted in a frameshift in codon 150 and produced an aberrant protein consisting of 184 residues. This is the first report of a mutation, in this gene, resulting in isolated congenital cataract.

Glial fibrillary acidic protein mutations in infantile, juvenile, and adult forms of Alexander disease

Alexander disease is a progressive, usually fatal neurological disorder defined by the widespread and abundant presence in astrocytes of protein aggregates called Rosenthal fibers. The disease most often occurs in infants younger than 2 years and has been labeled a leukodystrophy because of an accompanying severe myelin deficit in the frontal lobes. Later onset forms have also been recognized based on the presence of abundant Rosenthal fibers. In these cases, clinical signs and pathology can be quite different from the infantile form, raising the question whether they share the same underlying cause. Recently, we and others have found pathogenic, de novo missense mutations in the glial fibrillary acidic protein gene in most infantile patients examined and in a few later onset patients. To obtain further information about the role of glial fibrillary acidic protein mutations in Alexander disease, we analyzed 41 new patients and another 3 previously described clinically, including 18 later onset patients. Our results show that dominant missense glial fibrillary acidic protein mutations account for nearly all forms of this disorder. They also significantly expand the catalog of responsible mutations, verify the value of magnetic resonance imaging diagnosis, indicate an unexpected male predominance for the juvenile form, and provide insights into phenotype–genotype relations. Ann Neurol 2005;57:310–326

Altered aggregation properties of mutant γ-crystallins cause inherited cataract

Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non‐neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered γ‐crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of γ‐crystallin mutation, large inclusions containing the altered γ‐crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid‐detecting dye, Congo red. In vitro, recombinant mutant γB‐crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild‐type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid‐like inclusions. The mutant γ‐crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.

Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580

Stress‐activated protein kinase 2a, also called p38, is inhibited by SB 203580 and this drug has been used widely to implicate this enzyme in the regulation of many physiological processes. Here, we introduce a novel method of general application, which can be used to establish whether the effects of SB 203580 are mediated via inhibition of stress‐activated protein kinase 2a/p38 or whether they result from ‘non‐specific’ effects. Four events thought to occur upon activation of stress‐activated protein kinase 2a/p38 have been established unequivocally. These are the activation of mitogen‐activated protein kinase‐activated protein kinase‐2 and mitogen‐ and stress‐activated protein kinase‐1 and the phosphorylation of their presumed substrates, heat shock protein 27 and the transcription factor cyclic AMP response element binding protein, respectively. In contrast, the SB 203580‐induced activation of c‐Raf is independent of stress‐activated protein kinase 2a/p38 inhibition.

In vitro studies on the assembly properties of the lens proteins CP49, CP115: Coassembly with α-crystallin but not with vimentin

A rapid one-step purification procedure for CP49, an intermediate filament protein found in the lens, is described using reverse-phase HPLC. This protein is one of the major intermediate filament proteins of the lens fibre cells and is found in both the water insoluble fraction (WIF) and the water soluble fraction (WSF) of the lens. In order to better understand the physiological role of CP49 in lens transparency we have purified CP49 from both compartments and compared the in vitro assembly characteristics of both by electron microscopy and sedimentation assays. Our studies showed that CP49, when mixed with another lens intermediate filament protein, CP115, forms 10 nm intermediate filaments. Vimentin, another intermediate filament protein found in the lens, was unable to coassemble with CP115, thus demonstrating the specificity of the interaction of CP49 with CP115. CP49 isolated from either the WIF or the WSF formed 10-nm filaments with CP115 and indicated that CP49 from both these lens cell compartments had similar in vitro assembly characteristics. This also suggested that the post-translational modifications observed for CP49 from the different compartments was of little apparent consequence to filament formation. The inability to reconstitute beaded filaments from CP49 and CP115 suggested that other lens proteins may be needed in the reconstitution assay before these lens specific cytoskeletal elements could be repolymerised from their purified protein components. CP49 and CP115 were therefore assembled in the presence of alpha-crystallins and a beaded filament structure was observed as has been seen with type III intermediate filament proteins assembled with alpha-crystallins.

Functions of the intermediate filament cytoskeleton in the eye lens

Intermediate filaments (IFs) are a key component of the cytoskeleton in virtually all vertebrate cells, including those of the lens of the eye. IFs help integrate individual cells into their respective tissues. This Review focuses on the lens-specific IF proteins beaded filament structural proteins 1 and 2 (BFSP1 and BFSP2) and their role in lens physiology and disease. Evidence generated in studies in both mice and humans suggests a critical role for these proteins and their filamentous polymers in establishing the optical properties of the eye lens and in maintaining its transparency. For instance, mutations in both BFSP1 and BFSP2 cause cataract in humans. We also explore the potential role of BFSP1 and BFSP2 in aging processes in the lens.

Expression of individual lamins in basal cell carcinomas of the skin

In this study we used a unique collection of type specific anti-lamin antibodies to study lamin expression patterns in normal human skin and in skin derived from patients with basal cell carcinomas (BCCs). Lamin expression in serial sections from frozen tissue samples was investigated by single and double indirect immunofluorescence. In normal skin, lamin A was expressed in dermal fibroblasts and in suprabasal epithelial cells but was absent from all basal epithelial cells. Lamin C was expressed in dermal fibroblasts, suprabasal epithelial cells and a majority of basal epithelial cells. However, lamin C was not expressed in quiescent basal epithelial cells. Lamin B1was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2was expressed in all cell types in normal skin. Lamin expression was also investigated in a collection of 16 BCCs taken from a variety of body sites. Based upon patterns of lamin expression the BCCs were classified into four groups: A-negative (10/16 tumours), C-negative (5/16 tumours), A/C-negative (1/16 tumours) and A/B2-negative (1/16 tumours). Lamin expression was also compared to cell proliferation index by staining serial sections with the proliferation marker Ki67. 9/10 of the lamin A negative tumours were highly proliferative, whereas 4/5 of the lamin C negative tumours were slow growing. Thus as a general rule absence of lamin A was correlated with rapid growth within the tumour, while absence of lamin C was correlated with slow growth within the tumour. Our data supports the hypothesis that lamin A has a negative influence on cell proliferation and its down regulation may be a requisite of tumour progression.

Cytoskeletal Competence Requires Protein Chaperones

The cytoskeleton is the internal structure of the cell that makes diverse cellular functions possible. These structures are extremely dynamic, undergoing continual remodelling within the cytoplasm and requiring continual change in the protein-protein interactions as part of their function. It is therefore not surprising that cytoskeletal proteins require the attention of protein chaperones at all stages of their life. Initially, chaperonins ensure the nascent chains of actin and tubulin fold correctly as they emerge from the ribosome and in this review, the role of the small heat shock proteins (sHSPs) in further cytoskeletal function will be discussed. Chaperones are most certainly important components in the birth and life of the cytoskeleton.

Human keratin 8 mutations that disturb filament assembly observed in inflammatory bowel disease patients

We have identified miss-sense mutations in keratin 8 in a subset of patients with inflammatory bowel disease (Crohn disease and ulcerative colitis). Inflammatory bowel diseases are a group of disorders that are polygenic in origin and involve intestinal epithelial breakdown. We investigated the possibility that these keratin mutations might contribute to the course of the disease by adversely affecting the keratin filament network that provides mechanical support to cells in epithelia. The mutations (Gly62 to Cys, Ile63 to Val and Lys464 to Asn) all lie outside the major mutation hotspots associated with severe disease in epidermal keratins, but using a combination of in vitro and cell culture assays we show that they all have detrimental effects on K8/K18 filament assembly in vitro and in cultured cells. The G62C mutation also gives rise to homodimer formation on oxidative stress to cultured intestinal epithelial cells, and homodimers are known to be polymerization incompetent. Impaired keratin assembly resulting from the K8 mutations found in some inflammatory bowel disease patients would be predicted to affect the maintenance and re-establishment of mechanical resilience in vivo, as required during keratin cytoskeleton remodeling in cell division and differentiation, which may lead to epithelial fragility in the gut. Simple epithelial keratins may thus be considered as candidates for genes contributing to a risk of inflammatory bowel disease.