Our previous studies showed that recombinant £]-bungarotoxin B chain exhibited membrane-damaging activity. Given that membrane-damaging activity is crucial for bactericidal effect of antibacterial peptide, the causal relationship between membrane-damaging activity and antibacterial action of B chain was performed in this study. £]-bungarotoxin B chain exhibited a growth inhibition on Escherichia coli (Gram-negative bacteria), but marginally displayed bactericidal effect on Staphylococcus aureus (Gram-positive bacteria). Destabilization of lipopolysaccharide (LPS) layer and inhibition of lipoteichoic acid (LTA) biosynthesis on cell wall increased bactericidal effect of B chain on E. coli and S. aureus. B chain induced leakage and fusion of bacterial membrane-mimicking liposomes. Compared with LPS, LTA notably suppressed membrane-damaging activity and fusogenicity of B chain. B chain showed similar binding affinity with LPS and LTA. Circular dichroism measurement revealed that LPS- and LTA-binding differently induced conformational change of B chain. Taken together, our data indicate that antibacterial action of B chain is related to its ability to induce membrane permeability and fusogenicity, and suggest that LTA- and LPS- induced conformational change of B chain affect membrane-damaging activity, fusogenicity and antibacterial activity of B chain.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 238-308).

αB-crystallin is a small heat-shock chaperone protein (sHSP) required for the homeostasis of multiple tissues including eye lens, retina, heart and brain. Correspondingly, mutation or altered levels of αB-crystallin are associated with multiple degenerative diseases including cataract, retinal degeneration, cardiomyopathy and Lewy body disease. Based on its wide-ranging importance understanding the protective and homeostatic properties of α B-crystallin is critical for understanding degenerative diseases and could lead to the development of therapies to treat these diseases. αB-crystallin is localized to the mitochondria suggesting a direct effect on mitochondrial function. My thesis work has examined those molecular pathways required for translocation of αB-crystallin to the mitochondria and to identify the downstream pathways controlled by mitochondrial translocation of αB-crystallin that could be important for cellular protection and differentiation. My results point to a novel role of αB-crystallin in regulation of key apoptotic pathways that mediate the balance between cell survival and differentiation. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

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.

The major obstacle many promising drugs struggle to overcome is the barrier imposed by the outer cell membrane. In addition to technologies such as liposomes and cell-penetrating peptides, more attention is being given to the class of proteins known as lectins to deliver therapeutic and antigenic proteins to the interiors of cells. Lectins bind to but do not modify sugars, and provide an efficient route to endocytosis. The galactose/N-acetyl-galactosamine specific lectin ricin B-chain (RTB) is especially attractive in possibly fulfilling a carrier role due to its well-characterized endocytotic trafficking and its efficacy over a wide range of cell types. By producing RTB recombinantly in plants it is possible to create a fully active, non-toxic carrier that does not rely on the processing of large amounts of toxic material (e.g. castor bean). Payload molecules such as small molecules and proteins can be attached to RTB via chemical conjugation at primary amine groups, without the loss of lectin or uptake activities. The biotin/streptavidin interaction and direct genetic fusion of polypeptides also provide efficient mechanisms for the attachment of payload proteins to RTB. An immunoglobulin domain-based scaffolding mechanism bridges modified RTB and payload proteins when co-expressed in Agrobacterium-infiltrated plant leaves. Carrier and payload proteins expressed in plants and E. coli, respectively, and purified independently are not able to assemble into an efficient carrier/payload arrangement. These findings show that plant cells are able to correctly produce the two components of the carrier/payload system and assemble them into an efficient and flexible capture and carry technology. / Ph. D.