No description available.

Masseter muscle is one of the major muscles of mastication, and is comprised of actin and myosin myofibrils organized into sarcomeric contractile units. Structurally, sarcomeres are repeating portions of myofibrils between neighboring Z-lines (a.k.a. Z-disc, Z-band). The Z-line or Z-disc is composed of non-contractile proteins that provide mechanical stability to the sarcomere. One of the proteins of Z-disc is alpha-B-crystallin, a protein product of the gene CRYAB. Together with several other proteins of the Z-disc, CRYAB gene has been found to be up-regulated in Actn3 knock-out mice. In addition, CRYAB is suspected to be a pain mediator gene, having similar structure and function to CRYAA (alpha,A-crystallin) identified as one of the candidate genes from the Pain Research Panel, previously investigated in the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) Study. Finally, in a microarray of global gene expression CRYAB was increased in subjects with facial asymmetry. We have examined CRYAB expression in masseter muscle of 64 orthognathic surgery patients to determine associations with skeletal malocclusions. Salivary DNA was genotyped for a single nucleotide polymorphism (SNP) for ACTN3 (rs1815739) and masseter muscle RNA isolated from an orthognathic surgery patient population. These genotyping and expression data have been used to identify differences in CRYAB expression in sub-groups of our patient population with Class II and III, normal, open and deep bite malocclusions who are null for ACTN3. In addition, we evaluated expression levels of CRYAB in patients with TMD-related myalgia. We found that relative quantities of CRYAB expression differed very significantly between sexes (p=0.005). ANOVA comparison between all subjects with and without TMD-myalgia indicated that males with TMD-myalgia had significantly greater (p<0.02) expression than other groups. An unpaired t-test showed that with TMD-related myalgia, CRYAB expression was significantly higher (p=0.03) in males than in females. ANOVA comparison between sexes with Class II and Class III malocclusions showed greater expression of CRYAB (p=0.005) in males with Class II. Expression was likewise greater in males with Class III malocclusion than in females with Class III (p<0.01). Among different age groups, subjects 25 years of age or younger had significantly (p value=0.025) increased expression of CRYAB gene. There were no significant differences for genotypes or facial asymmetry. / Oral Biology

Purpose: To investigate species differences in the distribution and localization of alpha B-crystallin (ABC) in the normal retina and optic nerve head region, and to describe changes during outer and inner retina degeneration. Material and methods: Animals studied included mice, rats, cats, pigs, cows, and monkeys. Sections of the optic nerve and central retina were labeled with antibodies against ABC and glial fibrillary acidic protein (GFAP). Results: ABC was located in astrocytes and Muller cells with different intensities. During outer retina degeneration (dystrophic rat and Abyssinian cat), only late stages showed an increase in ABC in the retina and optic nerve head. Inner retina degeneration in the glaucoma mouse model showed no increase of ABC. In the monkey glaucoma model, only the innermost layer of the optic nerve head showed increased labeling for ABC. Conclusions: The distribution of ABC is species dependent and is (excluding the mouse) present in the nerve fiber layer of the retina and in the optic nerve head (localization of astrocytes). Chronic retinal degeneration does not necessarily lead to an over-expression of ABC. While in outer retinal degeneration induction was predominantly present in late stages, pressure-induced glaucoma led to a specific increase in ABC already in early stages indicating a local stress-response in this region.

No description available.

Small Heat Shock Proteins (sHSP) are critical molecular chaperones that function to maintain protein homeostasis (proteostasis) and prevent the aggregation of other proteins during cellular stress. Any disruption in the process of proteostasis can lead to prevalent diseases ranging from cancer and cataract to cardiovascular and Alzheimer's disease. CryAB (αB-crystallin, HspB5) is one of ten known human sHSP that is abundant in the lens, skeletal, and cardiac muscle. This protein is required for cardiac function and muscle cell integrity. When the cell experiences physiological stress, including heat shock, CryAB moves to the cytoskeleton to act as a chaperone and prevent aggregation of its protein clientele. This research is designed to investigate the molecular role of CryAB in cell proteostasis through the identification of putative protein clientele and chaperone activity analysis. We have identified over twenty CryAB-binding partners through combined yeast two-hybrid (Y2H) and co-purification approaches, including interactions with myofibril proteins. Previously reported disease-associated CryAB missense variants were analyzed in comparison to wild type CryAB through Y2H binding assays. The characterization of the similarities and differences in binding specificities of these variants provide a foundation to better understand the chaperone pathways of CryAB and how these changes in molecular function result in the development of disparate diseases such as cataract, cancer, and various myopathies.

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs), where it has been shown to have potent anti-apoptotic properties. The mechanism by which cryAB prevents apoptosis has not been fully characterized. Therefore, I was interested in elucidating its protective mechanism in CMs. I identified its sub-cellular localization and its binding interactors following H2O2 exposure. I found that cryAB is found in the cytosol under control conditions and that following H2O2 exposure it becomes phosphorylated and translocates to the mitochondria. CryAB silencing resulted in increased apoptosis levels in CMs. Co-immunoprecipitation revealed an apparent increased interaction of cryAB and PcryAB with mitochondrial VDAC, caspase 12 and uncleaved caspase 3 in stressed hearts relative to controls. These results suggest that the cardio-protective effects of cryAB are mediated by its translocation to the mitochondria and its interaction with VDAC, caspase 12 and caspase 3 following exposure to H2O2.

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs), where it has been shown to have potent anti-apoptotic properties. The mechanism by which cryAB prevents apoptosis has not been fully characterized. Therefore, I was interested in elucidating its protective mechanism in CMs. I identified its sub-cellular localization and its binding interactors following H2O2 exposure. I found that cryAB is found in the cytosol under control conditions and that following H2O2 exposure it becomes phosphorylated and translocates to the mitochondria. CryAB silencing resulted in increased apoptosis levels in CMs. Co-immunoprecipitation revealed an apparent increased interaction of cryAB and PcryAB with mitochondrial VDAC, caspase 12 and uncleaved caspase 3 in stressed hearts relative to controls. These results suggest that the cardio-protective effects of cryAB are mediated by its translocation to the mitochondria and its interaction with VDAC, caspase 12 and caspase 3 following exposure to H2O2.

sHSPs maintain partially denaturing substrates in a soluble sHSP-substrate complex. The heterogeneous interaction between sHSPs and substrate within the complex has prevented a detailed study of the mechanism of sHSP substrate protection. Here, purified sHSPs and heat sensitive substrates were used to investigate the mechanism of sHSP chaperone action. Results presented provide new insights into how sHSPs recognize substrates, the architecture of the sHSP-substrate complex and factors contributing to chaperone efficiency.Direct evidence defining the role of the sHSP N-terminal arm and alpha crystallin domain in sHSP-substrate interactions is limited. A photoactivatable probe was site- specifically incorporated into PsHsp18.1, and cross-linking to substrate in sHSP-substrate complexes was quantified. The structurally flexible N-terminal arm of PsHsp18.1 makes strong contacts with both substrates tested, however differences in interaction were seen in the conserved alpha crystallin domain. Regions on the sHSP showing the strongest cross-links to substrates are buried within the dodecamer, supporting the model that the sHSP oligomer undergoes rearrangement or dissociation prior to substrate interactions.The arrangement of sHSPs and substrates whithin the complex is poorly defined. Limited proteolysis and chemical modification was combined with mass spectrometry to probe the sHSP-substrate complex using multiple sHSPs and substrates. This analysis reveals that a similar partially-denatured form of substrate is protected in the complex irrespective of sHSP identity. Further, sHSP in the complex is protected from proteolysis for a longer time compared to free sHSP. These data suggest that sHSPs and substrate are distributed both internally and on the periphery of the sHSP-substrate complex.Exact properties of the sHSP N-terminal arm contributing to protection are poorly defined. A molecular dynamics (MD) study was designed to test the hypothesis that the N-terminal arm could assume multiple conformations that can readily interact with denaturing substrates. Preliminary data suggest that at increased temperatures amino acids in the N-terminal arm form specific clusters which could act as substrate interaction sites. MD simulations, mutagenesis and altering the kinetics of substrate aggregation suggest that the conformational space occupied by the N-terminal arm at increased temperatures, along with flexibility and rate of substrate aggregation contribute to differences in chaperone efficiency.

The muscle fibre cytoskeleton of skeletal and heart muscle cells is composed mainly of intermediate filaments (IFs), that surround the myofibrils and connect the peripheral myofibrils with the sarcolemma and the nuclear membrane. Desmin is the first muscle specific IF protein to be produced in developing muscles and is the main IF protein in mature muscles. In skeletal muscle, desmin is particularly abundant at myotendinous and neuromuscular junctions. In the heart an increased amount of desmin is found at intercalated discs and in Purkinje fibres of the conduction system. Interactions between the IFs themselves, and between IFs and other structures such as Z-discs and the sarcolemma, are mediated by intermediate filament associated proteins (IFAPs). A transgenic mice model, which lacks the desmin gene have been developed to study the function of desmin. In these mice, morphological abnormalities are observed in both heart and skeletal muscles. Similar defects have been observed in human myopathies, caused by different mutations in the desmin gene. In the present thesis, skeletal and heart muscles of both wild type and desmin knock-out (K/O) mice have been investigated. Furthermore the cytoskeletal organisation in skeletal muscles from human controls and from a patient with desmin myopathy was examined. In the desmin K/O mice, no morphological alterations were observed during embryogenesis. These mice postnatally developed a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. Ruptures of the sarcolemma appear to be the primary event leading to muscle degeneration and fibrosis both in cardiac and affected skeletal muscles. In the heart the muscle degeneration gave rise to calcifications, whereas in skeletal muscles regeneration of affected muscle was seen. In mature wild type mice, the IF proteins synemin and paranemin, and the IFAP plectin were present together with desmin at the myofibrillar Z-discs, the sarcolemma, the neuromuscular junctions and the myotendinous junctions. Nestin was only found in these junctional regions. In desmin K/O mice, all four proteins were detected at neuromuscular and myotendinous junctions. The normal network of synemin and paranemin were not observed, whereas the distribution of plectin was preserved. In normal human muscles, synemin, paranemin, plectin and αB-crystallin were colocalised with desmin in between the myofibrils, at the sarcolemma and at myotendinous and neuromuscular junctions. In the human desmin myopathy, the distribution of desmin varied considerably. A normal pattern was seen in some fibres areas, whereas other regions either contained large subsarcolemmal and intermyofibrillar accumulations of desmin or totally lacked desmin. Nestin, synemin, paranemin, plectin and αB-crystallin also exhibited an abnormal distribution. They were often aggregated in the areas that contained accumulations of desmin. In cultured satellite cells from the patient, a normal network of desmin was present in early passages, whereas aggragates of desmin occurred upon further culturing. In the latter, also the nestin network was disrupted, whereas vimentin showed a normal pattern. αB-crystallin was only present in cells with a disrupted desmin network. Plectin was present in a subset of cells, irrespective of whether desmin was aggregated or showed a normal network. From the present study it can be concluded that an intact desmin network is needed to maintain the integrity of muscle fibres. Desmin may be an important component in the assembly of proteins, which connect the extrasarcomeric cytoskeleton with the extracellular matrix.

Le gène CG4533 (l (2) efl, dcryAB) de la drosophile est un orthologue de la cristalline αB des vertébrés, qui code une petite protéine de choc thermique (sHsp). L'activité la plus importante des sHsps est de lier des protéines et de les protéger contre l'agrégation, empêchant l'accumulation de protéines partiellement dénaturées dans les cellules musculaires. La cristalline αB est également impliquée dans la phase initiale de différenciation du muscle squelettique. Il a été démontré que la cristalline αB joue un rôle clé dans la différenciation musculaire, et sa forme mutée est impliquée dans des desminopathies humaines. Mes résultats ont révélé que le gène dcryAB est spécifiquement exprimé dans les muscles des parois larvaires et que le profil d 'expression de dCryAB rappelle la localisation de l' orthologue αB-cristallin humain. En utilisant les anticorps polyclonaux générés, on a trouvé que la protéine dCryAB était abondamment exprimée dans tous les muscles de la paroi des larves, qu'elle s'accumulait dans une zone périnucléaire et présentait un profil strié au niveau des lignées M et Z. Pour évaluer le rôle de dcryAB dans le développement musculaire, l'atténuation des gènes spécifiques au muscle médiée par l'ARNi a été pratiquée. Le knockdown dcryAB conduit à des défauts importants dans la morphologie musculaire. La majorité des larves observées présentaient des défauts d'organisation des sarcomères, caractérisés par un motif irrégulier et flou des lignes Z dans les grands segments musculaires et un plus petit nombre de noyaux musculaires. Ce schéma sarcomérique aberrant était souvent associé à une scission musculaire entraînant une altération de l'attachement musculaire ou à la perte du muscle affecté. Les analyses ultrastructurales ont confirmé l'organisation altérée des sarcomères, révélant également des mitochondries à crêtes mitochondriales à peine visibles et une quantité accrue de glycogène entre les myofilaments. De plus, l'atténuation du dcryAB spécifiquement dans le mésoderme a conduit à une altération de la fonctionnalité des muscles larvaires et a affecté la durée de vie de la drosophile, ce qui implique le rôle du dcryAB dans le développement musculaire. Les études présentées ont également révélé que, dans les muscles larvaires de la drosophile, l'anticorps anti-vimentine de souris détectait une protéine co-localisée avec dCryAB dans la lignée Z et était présente dans la zone nucléaire. Je crois que Drosophila exprime des protéines qui correspondent à la troisième classe de filaments intermédiaires de vertébrés, qui partagent une distribution intracellulaire similaire avec leurs homologues vertébrés. Des approches co-IP ont confirmé que dCryAB interagit avec la protéine de type vimentine comme l'αB-cristalline interagit avec la desmine. Ainsi, il est attendu que la protéine de type vimentine puisse avoir des propriétés des protéines de filaments intermédiaires de troisième classe, renfermant de la vimentine et de la desmine. Des analyses effectuées suggèrent que dCryAB assure l'intégrité structurale des muscles somatiques en interagissant avec les protéines IF potentielles. Chez l'homme, la substitution de R120G dans l'αB-cristalline conduit à la perte de son activité chaperon IF et induit des agrégations de desmine dans les muscles, provoquant une myopathie liée à la desmine (DRM). L'avantage de la conservation de l'αB-cristalline a été pris pour tester si dCryAB muté Drosophila affiche des propriétés similaires à celle de son homologue vertébré. J'ai trouvé que l'expression musculaire spécifique de dCryABR120G muté a provoqué la formation d'agrégats intracellulaires contenant la protéine de type vimentine ainsi que dCryABR120G. Ces symptômes ont conduit à une faiblesse musculaire caractéristique des patients avec DRM. Mes études ont révélé que le dCryABR120G imite les effets de la mutation dans l'αB-cristalline humaine, donc Drosophila peut représenter un système modèle approprié pour étudier la DRM. / The Drosophila CG4533 (l(2)efl, dcryAB) gene is an orthologue of vertebrate αB-crystallin, which encodes a small heat shock protein (sHsp). The most prominent activity of sHsps is binding proteins and protecting them from aggregation, preventing the accumulation of partially denatured or improperly folded proteins in muscle cells. αB-­crystallin is also implicated in the initial phase of skeletal muscle differentiation. It was demonstrated that αB-­crystallin plays a key role in muscle differentiation and its mutated form is involved in human desminopathies. My results revealed that dcryAB gene is specifically expressed in larval body wall muscles and the dCryAB expression pattern was reminiscent of the localisation of its human orthologue αB-­crystallin. Using the generated polyclonal antibodies it was found that dCryAB protein was abundantly expressed in all larval body wall muscles, it was accumulated in a perinuclear area and displayed a striated pattern at the level of M-­ and Z-­lines. To assess the dcryAB role in muscle development RNAi-­mediated muscle-­specific gene attenuation was applied. The dcryAB knockdown led to formation of muscles characterised by significant defects in muscle morphology. The majority of the observed larvae exhibited defects in sarcomeric organisation, characterised by an irregular, fuzzy pattern of Z-­lines in large muscle segments and smaller number of muscle nuclei. This aberrant sarcomeric pattern was often associated with muscle splitting leading to an altered muscle attachment or to the loss of the affected muscle. Ultrastuctural analyses confirmed altered sarcomeres organisation, revealing also mitochondria with barely visible mitochondrial crests and increased amount of glycogen between myofilaments. Moreover, dcryAB attenuation specifically in mesoderm led to impaired functionality of larval muscles and affected Drosophila life span, implicating dcryAB role in muscle development. Presented studies also revealed that in Drosophila larval muscles mouse anti-­vimentin antibody detected a protein which co-­localised with dCryAB in the Z-­line and was present in the nuclear area. I believe that Drosophila expresses proteins that correspond to the third class of vertebrate intermediate filaments, which share a similar intracellular distribution with their vertebrate counterparts. Using co-­IP approaches it was confirmed that dCryAB interacts with the vimentin-­like protein like αB-­crystallin interacts with desmin. Thus it is expected that the vimentin-­like protein may have properties of the third class intermediate filament proteins, enclosing vimentin and desmin. Performed analyses suggest that dCryAB ensures structural integrity of somatic muscles by interacting with potential IF proteins. In human, R120G substitution in αB-­crystallin leads to the loss of its IF chaperone activity and induces aggregations of desmin in muscles, causing desmin-­related myopathy (DRM). The advantage of the αB-­crystallin conservation has been taken to test whether Drosophila mutated dCryAB displays similar properties as its vertebrate counterpart. I found that muscle-­specific expression of mutated dCryABR120G caused formation of intracellular aggregates containing the vimentin-­like protein as well as dCryABR120G. These symptoms led in consequence to muscle weakness, which is characteristic for patients with DRM. My studies revealed that mutated dCryABR120G mimics effects of mutation in human αB-­crystallin, therefore Drosophila may represent a suitable model system to study DRM.