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Cell specific transcriptional regulation of the smooth muscle [alpha]-actin gene promoter : two M-CAT elements have differences in functionalactivity and binding properties in smooth muscle versus non-smooth muscle cells /Swartz, Ellen Ashley. January 1997 (has links)
Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: M-CAT motifs of the SM [alpha]-actin gene. Includes bibliographical references (90-100). Also available online through Digital Dissertations.
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In vitro Studies of Myofibers and Their Use in Analyzing the Differential Dynamics and Properties of α-Actinin IsoformsHsu, Cynthia Pu-Chun 04 June 2015 (has links)
Skeletal muscle is a highly organized tissue that requires cooperation of many different structures and components for proper function. We explored the use of a flexor digitorum brevis (FDB) myofiber culture system to better model highly differentiated aspects of skeletal muscle in an in vitro system. Indirect immunofluorescence of FDB myofibers allowed us to better determine the subcellular localization of KLHL41, a new nemaline myopathy (NM) gene product, to ER-like subdomains of the sarcoplasmic retiuculum. By comparing FDB myofibers from wild type and myotubularin knockout mice with X-linked myotubular myopathy (XLMTM), we were also able to analyze satellite cell populations, showing that the knockout mice suffered a marked decrease in associated myogenic satellite cells. This supports concurrent data from our lab indicating a disease progression-related increase in apoptosis and a decrease in satellite cell proliferation in XLMTM.
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Ca²⁺ and phosphoinositides regulations in α-actinin -4 F-actin binding.Chen, Huang-Hui January 2008 (has links)
α-actinin-4 is a non-muscle isoform of α-actinin that belongs to the spectrin superfamily. It comprises three functional regions: an N-terminal actin-binding region that consists of two calponin homology (CH) domains, a central region that consists of four copies of the spectrin-like repeat domain and a C-terminal calmodulin-like domain that is predicted to bind Ca²⁺. α-actinin-4 is organised as an antiparallel homodimer formed by the interaction of four spectrin-like repeats between the two monomers, giving a rod-like shape, with actin binding regions at both ends. α-actinin-4 is an abundant actin-bundling protein, which provides a direct link between actin filaments and integrins, and is believed to play an important role in stabilising cell shape and adhesion and regulating cell migration. It also acts as a tumor suppressor and influences the metastatic potential and invasiveness in human cancers. A cluster of three actin binding motifs have been identified in the CH domains (2X CH) from other members of the spectrin superfamily, utrophin and dystrophin. Two of them reside in the CH1 domain and the third resides in the first α-helix of the CH2 domain. In addition, a PIP2 binding site has been mapped on a region adjacent to actin-binding site-3. These observations imply the F-actin binding activity would be regulated by phosphoinositides. Five mutations of α-actinin-4, K122N, an alternative splice variant, K255E, T259I and S263P, have been reported to be involved in three human diseases, non-small lung cancer (NSCLC), small cell lung cancer (SCLC) and focal segmental glomerulosclerosis (FSGS). The mutation site within these mutants is located on the actin binding region. Therefore, the actin binding region is presumed to be associated with the progression of human disease. The aims of this thesis focused on the regulation of the F-actin binding activity of α-actinin-4 by phosphoinositides (PIP2 and PIP3), the calmodulin-like domain and Ca²⁺ , determination of the three-dimensional structure of the CH2 domain in solution and identification of the phosphoinositide binding site on the CH2 domain. In order to investigate the F-actin binding activity quantitatively, a novel in vitro F-actin binding assay (solid phase) was established to replace the semi-quantitative actin bundling assay. Using this novel solid phase F-actin binding assay, Ca²⁺ was shown to enhance the F-actin binding activity of α-actinin-4 in a concentration-dependent manner. The presence of 10 mM Ca²⁺ results in a two-fold increase in the F-actin binding activity. Both PIP2 and PIP3 inhibited the F-actin-binding activity of α-actinin-4 in a concentration-dependent manner with an approximate IC₅₀ of 75 and 45 μM, respectively. In order to characterise how phosphoinositides regulated the F-actin binding activity of α-actinin-4, the solution structure of α-actinin-4 CH2 domain was determined and the phosphoinositide binding residues within the CH2 domain were identified using NMR spectroscopy. The solution structure of α-actinin-4 CH2 domain contained six α-helices and was similar to that of other spectrin superfamily members. The strategy used in identification of the phosphoinositide binding site was an NMR-based 2D ¹H-¹⁵N HSQC ligand titration assay to replace the traditional semi-quantitative protein-lipid overlay assay. Using the NMR-based ligand titration assay, the recognition site for the inositol head group resides in residues Trp 172, Tyr 265 and His 266 and the binding region of acyl chains resides in the first α-helix structure which is one of the putative F-actin binding sites. In order to examine the interaction of phosphoinositides with this site, Y265A and H266E mutants of α-actinin-4 CH2 domain were generated using site-directed mutagenesis and verified the interaction with phosphoinositides and the inositol head group using an NMR-based ligand titration assay. These results confirmed the phosphoinositide binding site on the CH2 domain and residues, Tyr 265 and His 266, are critical for interacting with phosphoinositides. Wildtype and mutants (Y265A and H266E) of α-actinin-4 were expressed in mammalian cells as EGFP-fusion proteins. Wildtype α-actinin-4 was shown to be co-localised with focal adhesions and actin stress fibres. However, Y265A and H266E mutants of α-actinin-4 were co-localised with actin stress fibres but poorly co-localised with focal adhesions. Moreover, both Y265A and H266E mutants of α-actinin-4 were co-localised with actin in the cytoplasm rather than localised along the cell membrane after EGF stimulation for 30 minutes. These results suggested that PIP2 assists the co-localisation of α-actinin-4 with focal adhesions. Taken together, the results described in this thesis concluded that Ca²⁺ enhanced the F-actin binding activity of α-actinin-4 in vitro. However, phosphoinositides (PIP2 or PIP3) inhibited the F-actin binding activity in vitro. Moreover, the results described in this thesis provided a phosphoinositide binding site on α-actinin-4 CH2 domain. Binding to PIP2 is important to the localisation of α-actinin-4 in focal adhesions. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008
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PDZ-LIM domain proteins and α-actinin at the muscle Z-diskKlaavuniemi, T. (Tuula) 24 November 2006 (has links)
Abstract
The Z-disk is a sophisticated structure that connects adjacent sarcomeres in striated muscle myofibrils. α-Actinin provides strength to the Z-disks by crosslinking the actin filaments of adjacent sarcomeres. α-Actinin is an antiparallel homodimer, composed of an N-terminal actin binding domain (ABD), the central rod domain, and two pairs of C-terminal EF-hands. The PDZ-LIM domain proteins interact with α-actinin at the Z-disk. Of these proteins, only the actinin-associated LIM protein (ALP), Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) and C-terminal LIM protein (CLP36) have a ZASP/Cypher-like (ZM) motif consisting of 26-27 conserved residues in the internal region between the PDZ and LIM domains. The aim of this work was to understand the molecular interplay between the ZM-motif containing members of the PDZ-LIM proteins and α-actinin. To unveil the biological relevance of the interaction between the PDZ-LIM proteins and α-actinin, naturally occurring human ZASP/Cypher mutations were analyzed.
Two interaction sites were found between ALP, CLP36 and α-actinin using recombinant purified proteins in surface plasmon resonance (SPR) analysis. The PDZ domain of ALP and CLP36 recognized the C-terminus of α-actinin, whereas the internal regions bound to the rod domain. Further characterization showed that the ALP internal region adopts and extended conformation when interacting with α-actinin and that the ZM-motif partly mediated the interaction, but did not define the entire interaction area. ZASP/Cypher also interacted and competed with ALP in binding to the rod domain. The internal fragments containing the ZM-motif were important for co-localization of ALP and ZASP/Cypher with α-actinin at the Z-disks and on stress fibers. The absence of ALP and ZASP/Cypher in focal contacts indicates that other interacting molecules, for instance vinculin and integrin, may compete in binding to the rod in these areas or additional proteins are required in targeting to these locations. The co-localization of the ZASP/Cypher with α-actinin could be released by disrupting the stress fibers leading to an accumulation of α-actinin in the cell periphery, whereas ZASP/Cypher was not in these areas. This suggests that an intact cytoskeleton is important for ZASP/Cypher interaction with α-actinin. Earlier studies have shown that mutations in the ZASP/Cypher internal region are associated with muscular diseases. These mutations, however, did not affect ZASP/Cypher co-localization with α-actinin or the stability of ZASP/Cypher proteins.
The Z-disk possesses a stretch sensor, which is involved in triggering hypertrophic growth as a compensatory mechanism to increased workloads. α-Actinin is a docking site of molecules that are involved in hypertrophic signaling cascades mediated by calsarcin-calcineurin and protein kinase C (PKC) isoforms. The internal interaction site may be involved in targeting PKCs, which bind to the LIM domains of ZASP/Cypher, to the Z-disks. The similar location of the internal interaction site with calsarcin on the rod suggests that ZASP/Cypher, ALP and CLP36 may regulate calsarcin-mediated hypertrophic signaling.
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Human 36kda carboxyl terminal lim domain protein (HCLIM1): a novel lim domain protein that interacts with α-actinin 2. / CUHK electronic theses & dissertations collectionJanuary 1999 (has links)
Masayo Kotaka. / "May 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 179-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.
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Molecular characterization and evolution of alpha-actinin : from protozoa to vertebratesVirel, Ana January 2006 (has links)
<p>alpha-actinin is a ubiquitous protein found in most eukaryotic organisms. The ability to form dimers allows alpha-actinin to cross-link actin in different structures. In muscle cells alpha-actinin is found at the Z-disk of sarcomeres. In non-muscle cells alpha-actinin is found in zonula adherens or focal adhesion sites where it can bind actin to the plasma membrane.</p><p>alpha-actinin is the shortest member of the spectrin superfamily of proteins which also includes spectrin, dystrophin and utrophin. Several hypotheses suggest that alpha-actinin is the ancestor of this superfamily.</p><p>The structure of alpha-actinin in higher organisms has been well characterized consisting of three main domains: an N-terminal actin-binding domain with two calponin homology domains, a central rod domain with four spectrin repeats and a C-terminal calcium-binding domain. Data mining of genomes from diverse organisms has made possible the discovery of new and atypical alpha-actinin isoforms that have not been characterized yet.</p><p>Invertebrates contain a single alpha-actinin isoform, whereas most of the vertebrates contain four. These four isoforms can be broadly classified in two groups, muscle isoforms and non-muscle isoforms. Muscle isoforms bind actin in a calcium independent manner whereas non-muscle isoforms bind actin in a calcium-dependent manner.</p><p>Some of the protozoa and fungi isoforms are atypical in that they contain fewer spectrin repeats in the rod domain. We have purified and characterized two ancestral alpha-actinins from the parasite Entamoeba histolytica. Our results show that despite the shorter rod domain they conserve the most important functions of modern alpha-actinin such as actin-bundling formation and calcium-binding regulation. Therefore it is suggested that they are genuine alpha-actinins.</p><p>The phylogenetic tree of alpha-actinin shows that the four different alpha-actinin isoforms appeared after the vertebrate-invertebrate split as a result of two rounds of genome duplication. The atypical alpha-actinin isoforms are placed as the most divergent isoforms suggesting that they are ancestral isoforms. We also propose that the most ancestral alpha-actinin contained a single repeat in its rod domain. After a first intragene duplication alpha-actinin with two spectrin repeats were created and a second intragene duplication gave rise to modern alpha-actinins with four spectrin repeats.</p>
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Molecular characterization and evolution of alpha-actinin : from protozoa to vertebratesVirel, Ana January 2006 (has links)
alpha-actinin is a ubiquitous protein found in most eukaryotic organisms. The ability to form dimers allows alpha-actinin to cross-link actin in different structures. In muscle cells alpha-actinin is found at the Z-disk of sarcomeres. In non-muscle cells alpha-actinin is found in zonula adherens or focal adhesion sites where it can bind actin to the plasma membrane. alpha-actinin is the shortest member of the spectrin superfamily of proteins which also includes spectrin, dystrophin and utrophin. Several hypotheses suggest that alpha-actinin is the ancestor of this superfamily. The structure of alpha-actinin in higher organisms has been well characterized consisting of three main domains: an N-terminal actin-binding domain with two calponin homology domains, a central rod domain with four spectrin repeats and a C-terminal calcium-binding domain. Data mining of genomes from diverse organisms has made possible the discovery of new and atypical alpha-actinin isoforms that have not been characterized yet. Invertebrates contain a single alpha-actinin isoform, whereas most of the vertebrates contain four. These four isoforms can be broadly classified in two groups, muscle isoforms and non-muscle isoforms. Muscle isoforms bind actin in a calcium independent manner whereas non-muscle isoforms bind actin in a calcium-dependent manner. Some of the protozoa and fungi isoforms are atypical in that they contain fewer spectrin repeats in the rod domain. We have purified and characterized two ancestral alpha-actinins from the parasite Entamoeba histolytica. Our results show that despite the shorter rod domain they conserve the most important functions of modern alpha-actinin such as actin-bundling formation and calcium-binding regulation. Therefore it is suggested that they are genuine alpha-actinins. The phylogenetic tree of alpha-actinin shows that the four different alpha-actinin isoforms appeared after the vertebrate-invertebrate split as a result of two rounds of genome duplication. The atypical alpha-actinin isoforms are placed as the most divergent isoforms suggesting that they are ancestral isoforms. We also propose that the most ancestral alpha-actinin contained a single repeat in its rod domain. After a first intragene duplication alpha-actinin with two spectrin repeats were created and a second intragene duplication gave rise to modern alpha-actinins with four spectrin repeats.
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The Role of Ubiquitin C-Terminal Hydrolase L1 in Renal Function and Glomerular DiseaseBoisvert, Naomi January 2017 (has links)
Ubiquitin C-terminal hydrolase L1 is a deubiquitinating enzyme that salvages ubiquitin from substrates and maintains intracellular ubiquitin pools. While the role of ubiquitin C-terminal hydrolase L1 is well characterized in neurons, there is an increasing scope of evidence to suggest that ubiquitin C-terminal hydrolase L1 also plays a role in renal function and glomerular disease, however, its specific role in these settings remains incompletely elucidated. In the present thesis we explored the role of ubiquitin C-terminal hydrolase L1 in a mouse model of glomerular disease, ACTN4-associated focal segmental glomerulosclerosis and the role of ubiquitin C-terminal hydrolase L1 in renal function. Deletion of ubiquitin C-terminal hydrolase L1 in a mouse model of ACTN4-associated focal segmental glomerulosclerosis significantly improved indices of podocyte injury, a likely result of ubiquitin pool attenuation and sustained α-actinin-4 levels. However, global ablation of ubiquitin C-terminal hydrolase L1 in mice led to altered renal hemodynamics, namely glomerular hyperfiltration, most likely attributed to nerve dysfunction and loss of arterial resistance. Finally, mice lacking ubiquitin C-terminal hydrolase L1 exhibited perturbations in phosphate homeostasis as these showed evidence of hyperphosphatemia and phosphaturia, indicating altered renal phosphate balance. Altogether, these data show that while ubiquitin C-terminal hydrolase L1 plays a maladaptive role in glomerular disease, it also functions as a crucial regulator of renal hemodynamics and renal phosphate handling, suggesting that it may have distinct functions in diseased and non-diseased kidneys.
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Vieillissement musculaire : impact de la protéolyse intracellulaire calcium-dépendanteBrulé, Cédric 25 November 2009 (has links)
La sarcopénie ou perte involontaire progressive de la masse musculaire chez le sujet âgé s’accompagne de l’altération de nombreux phénomènes physiologiques comparables à ceux observés chez les myopathes. Le processus de régénération musculaire est très ralenti, les activités protéolytiques intracellulaires sont modifiées et de nombreuses fonctions cellulaires sont perturbées en raison d’un stress oxydatif incontrôlé. L’intervention des calpaïnes, protéases neutres calcium-dépendantes, dans les processus associés au développement, à la régénération et à l’intégrité du tissu musculaire est incontestable. Les calpaïnes apparaissent, en effet, comme des acteurs clefs des voies de transductions liées à la myogenèse, la prolifération et la survie cellulaire. Toutefois aucune étude permettant d’établir la relation vieillissement du tissu musculaire- activité calpaïne n’a été entreprise à ce jour. Le projet a donc pour but principal d’inventorier les signaux pro-sarcopéniques interagissant avec les calpaïnes et d’établir leurs relations avec la fonctionnalité des cellules satellites, le stress oxydant et l’apoptose. Nous avons mis en évidence une augmentation de l’expression/activité des calpaïnes durant le vieillissement musculaire chez le rat et identifié des partenaires des calpaïnes impliqués dans des fonctions physiologiques altérées durant la sarcopénie: homéostasie calcique, activité contractile, production d’ATP, régénération musculaire. Nous avons également montré que l’induction d’un stress oxydant entraîne l’activation des calpaïnes au cours de la prolifération des cellules satellites de façon corrélée à une augmentation de l’apoptose. D’une manière intéressante, un traitement préventif par un antioxydant naturel d’écorce de pin (Oligopin®) est capable de prévenir à la fois l’apoptose et l’activation des calpaïnes. L’ensemble de ces résultats suggère que le stress oxydant associé au vieillissement induirait des mécanismes calpaïno-dépendants responsables de l’altération de processus essentiels à la fonction musculaire. / Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition represents a major public health concern. Although sarcopenia is well documented, the molecular mechanisms of this condition still remain unclear. The calcium-dependent proteolytic system is composed of calcium dependent cystein-proteases named calpains. Calpains are involved in a large number of physiological processes such as muscle growth and differentiation, and pathological conditions such as muscular dystrophies. The aim of this study was to determine the involvement of the proteolytic system in the phenotype associated with sarcopenia by identify the key proteins (substrates or regulators) interacting with calpains during muscle aging and identify pro-sarcopenic signals after oxidative stress induction in satellite cells. Muscle aging was correlated with the up-regulation of calpain activity. Ryanodine receptor 1, ATP synthase subunit alpha and alpha actinin 3 appear as key partners of calpains during muscle aging. Such interactions suggest an implication of calpains in many processes altered during aging including cytoskeletal disorganisation, regulation of calcium homeostasis and mitochondrial dysfunction. Furthermore, oxidative stress induction led to an increase in the activity of calpains correlated to an increase in apoptosis of proliferating satellite cells. In a very interesting way, a preventive treatment with a commercial antioxidant (Oligopin®) prevented these effects. All these data suggest that oxidative stress coupled observed during muscle aging could lead to calpaïno-dependent mechanisms responsible for apoptosis and muscle dysorganisation.
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X-ray characterization of PaPheOH, a bacterial phenylalanine hydroxylaseEkström, Fredrik January 2003 (has links)
<p>Many human diseases are associated with the malfunction of enzymes in the aromatic amino acid hydroxylase family, e.g. phenylketonuria (PKU), hyperphenylalaninemia (HPA), schizophrenia and Parkinson's disease. The family of aromatic aminoacid hydroxylases comprises the enzymes phenylalanine hydroxylase (PheOH), tyrosine hydroxylase (TyrOH) and tryptophane hydroxylase (TrpOH). These enzymes require the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and atomic oxygen. In eukaryotes, the aromatic amino acid hydroxylases share the same organization with a N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain. Aromatic amino acid hydroxylases that correspond to the core catalytic domain of the eukaryotic enzymes are found in bacteria. The main focus of this thesis is the structural characterization of a phenylalanine hydroxylase from the bacterium Pseudomonas aeruginosa (PaPheOH). </p><p>To initiate the structural characterization, the active site environment was investigated with X-ray absorption spectroscopy (XAS). The experimental data support a model where the active site iron is coordinated by four oxygen atoms and two nitrogen atoms. We suggest that two water molecules, His121, His126 and Glu166 coordinates the active site iron. In this model, Glu166 provides two of the oxygen atoms in a bidentate binding geometry. EXAFS and XANES studies indicate that structural rearrangements are induced in the second and third coordination shells in samples of PaPheOH with BH4 and/or L-Phe. </p><p>The 1.6 Å X-ray structure of PaPheOH shows a catalytic core that is composed of helices and strands in a bowl-like arrangement. The iron is octahedrally coordinated, by two water molecules and the evolutionary conserved His121, His126 and Glu166 that coordinates the iron with bidentate geometry. The pterin binding loop of PaPheOH (residue 81-86) adopts a conformation that is displaced by 5-6 Å from the expected pterin binding site. Consistent with the unfavourable position of the pterin binding loop is the observation that PaPheOH has a low specific activity compared to the enzymes from human and Chromobacterium violaceum. </p><p>The second part of this thesis focus on the crystallization and structure determination of the actin binding domain of a-actinin (ABD). a-Actinin is located in the Z-disc of skeletal muscle were it crosslinks actin filaments to the filamentous protein titin. The ABD domain of a-actinin crystallizes in space group P21 with four molecules in the asymmetric unit. The structure of the ABD domain has been solved to a d-spacing of 2.0 Å. The two CH-domains of ABD is composed of 5 a-helices each. The a-helices fold into a closed compact conformation with extensive intramolecular contacts between the two domains.</p>
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