To investigate mechanisms responsible for the turnover of endogenous muscle protein, lysosomotropic proteinase inhibitors have been employed to elucidate the relative contributions of lysosomal and non-lysosomal degradation pathways functioning under varying nutritional states and for different classes of intracellular proteins. Proteolysis in cultured bovine aortic smooth muscle cells was measured as the percentage of ³H-phenylalanine released per hour from pre-labelled cellular proteins. To reduce background radioactivity, the intracellular ³H-phenylalanine pool was depleted by serial extraction at 37°C, effecting equilibration between the intracellular pool and the phenylalanine-free medium. Reutilization of labelled amino acids during subsequent incubation periods was minimized by the presence of excess non-labelled phenylalanine in the medium. ³H-phenylalanine was released at a constant rate of 1,5 % per hour for at least 4 h, from cells pre-labelled for 16 h ('long-lived' proteins). Leupeptin, an inhibitor of thiol proteinases including cathepsin 8, inhibited degradation by 12 %, whereas the general lysosomal inhibitors chloroquine and NH₄Cl inhibited degradation by 30 %, presumably the contribution by the lysosomal pathway. In the case of 'short-lived' proteins (pre-labelled for 1 hour), the initial degradation rate was 6,5% per hour, which rapidly declined, reaching the basal rate of 1,5 % after 4 h. Chloroquine and NH₄Cl reduced proteolysis by only 12-15% and leupeptin had no significant inhibition, consistent with the view that the majority of short-lived proteins a degraded by non-lysosomal pathways. Proteolysis rates of 'abnormal' proteins containing the arginine-analogue, canavanine, were found to be significantly elevated (80 %) over controls. Leupeptin had no significant inhibition, and chloroquine and NH₄Cl only reduced degradation by 12-16 %, showing that the rapid removal of 'abnormal' intracellular proteins proceeds mainly via extra-lysosomal mechanisms. Incubation of the cells under nutritional step-down conditions, increased the average degradation rate of long-lived proteins to 3% per hour, and chloroquine and NH₄Cl inhibited degradation by 55-60 %, indicating that the accelerated proteolytic condition is due to increased activity of the lysosomes. Nutritional deprivation did not increase the rate of degradation of short-lived proteins. The results were clarified by the parallel use of the well-characterized LDL degradation system in this cell type, known to occur almost exclusively via lysosomes. This allowed the effectiveness of lysosomotropic inhibitors to be tested. Chloroquine inhibited LDL degradation by over 90 % and NH₄Cl inhibited by 80-95 % in all cases. Other proteinase inhibitors such as chymostatin, pepstatin and the chloromethyl ketones were also tested, and of these chymostatin seemed to be the most valuable because of its additivity to the effect of chloroquine, indicating its selective inhibition of non-lysosomal degradative mechanisms. Incubations of smooth muscle cells under anoxic conditions or with metabolic inhibitors such as fluoride, azide and cyanide, resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of chloroquine, i.e. both lysosomal and non-lysosomal degradation pathways have some energy-dependence. The degradation of long-lived proteins appeared to be more sensitive to temperature than that of short-lived proteins, further indicating the activity of distinct proteolytic mechanisms for these two classes of intracellular proteins. Preliminary studies have indicated a role for Ca⁺⁺ in the regulation of proteolysis, since degradation rates were increased by elevated levels of Ca⁺⁺ in the extracellular medium. Inhibition of this increased proteolysis by leupeptin has indicated a role for a thiol proteinase, possibly Ca⁺⁺-activated neutral proteinase. In similar studies with cultured L8 skeletal muscle cells, an average proteolysis rate of 1,2 % per hour was found, which was increased by 50 % under nutritional step-down conditions. Once again, the lysosomal pathway was found to account for only about one-third of basal protein degradation but fully accounted for the increased proteolysis under nutrient deprivation. The degradation characteristics of intracellular smooth and skeletal muscle cell proteins was examined using double isotope labelling. It was found that large molecular weight proteins and glycoproteins tended to be degraded more rapidly than small proteins and non-glycoproteins. In smooth muscle cells, these correlations were markedly reduced or absent under the accelerated proteolysis associated with nutrient deprivation, possibly confirming the increased activity of the non-selective autophagic lysosomal pathway under these conditions. A similar loss of correlations was not so clearly seen for skeletal muscle cell proteins.

In order to understand the structural changes in myosin S1, fluorescence polarization and computational dynamics simulations were used. Dynamics simulations on the S1 motor domain indicated that significant flexibility was present throughout the molecular model. The constrained opening versus closing of the 50 kDa cleft appeared to induce opposite directions of movement in the lever arm. A sequence called the "strut" which traverses the 50 kDa cleft and may play an important role in positioning the actomyosin binding interface during actin binding is thought to be intimately linked to distant structural changes in the myosin's nucleotide cleft and neck regions. To study the dynamics of the strut region, a method of fluorescent labeling of the strut was discovered using the dye CY3. CY3 served as a hydrophobic tag for purification by hydrophobic interaction chromatography which enabled the separation of labeled and unlabeled species of S1 including a fraction labeled specifically at the strut sequence. The high specificity of labeling was verified by proteolytic digestions, gel electrophoresis, and mass spectroscopy. Analysis of the labeled S1 by collisional quenching, fluorescence polarization, and actin-activated ATPase activity were consistent with predictions from structural models of the probe's location. Although the fluorescent intensity of the CY3 was insensitive to actin binding, its fluorescence polarization was notably affected. Intriguingly, the mobility of the probe increases upon S1 binding to actin suggesting that the CY3 becomes displaced from interactions with the surface of S1 and is consistent with a structural change in the strut due to cleft motions. Labeling the strut reduced the affinity of S1 for actin but did not prevent actin-activated ATPase activity which makes it a potentially useful probe of the actomyosin interface. The different conformations of myosin S1 indicated that the strut is not as flexible as several other key regions of myosin as determined by the application of force constraints to elastic portions of the myosin structure.

Hoi-Yeung Li. / "May 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 151-168). / 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.

Graduation date: 1996

Graduation date: 1994

Thesis (Ph. D.)--University of California, San Diego, and San Diego State University, 2004. / Vita. Includes bibliographical references.

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Kinesiology and Health Science. / Typescript. Includes bibliographical references (leaves 71-78). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38823

Alternative splicing from a single gene or transcription from distinct genes are the two most important genetic mechanisms used to generate protein isoforms. This study concerns the ascidian Ciona intestinalis and the genetic mechanism used in this primitive chordate organism to produce differentially expressed isoforms of the muscle protein troponin I (TnI). / Through cDNA cloning and reverse-transcription PCR approaches, the mRNA sequences encoding two distinct Ciona ThI isoforms were determined. These mRNAs were found to be tissue-specifically expressed; one in the heart and the other in body-wall muscle. They encode long (229-residue) and short (182-residue) TnI protein isoforms homologous to vertebrate heart and skeletal muscle TnI isoforms, respectively. By sequence comparison, allelic polymorphism analysis, and gene structure determination, the two mRNAs were shown to be produced from a single gene by inclusion/exclusion of two serial internal exons. This tissue-specific alternative splicing mechanism contrasts with the vertebrate strategy of TnI isoform. production by differential transcription of a multigene family. This is the first reported case in which homologous tissue-specific protein isoforms are produced by non-homologous molecular mechanisms in related organismal lineages, implying that the molecular mechanism of isoform generation was entirely reworked in one of these lineages. An evolutionary scenario is presented that can account for such a reworking through a series of reasonable steps.

Sequence analysis of 5.4 kilobases (kb) of DNA upstream of the ATG initiation codon of the troponin I (TnI) gene of the ascidian Ciona intestinalis failed to identify a sequence corresponding to the first 16 nucleotides (nt) at the 5' end of the mature TnI mRNA. Database searching revealed that three additional and unrelated Ciona mRNAs contained the same, or almost identical, 16 nt sequence at the 5' -end. Reverse-transcriptase/polymerase chain reaction (RT-PCR) experiments proved that at least three additional mRNAs contain the same 5' -end sequence. These results suggested the possibility that the 5 ' end of the TnI, and other, Ciona mRNAs is derived by spliced leader (SL) trans-splicing. Also consistent with SL trans-splicing were Ciona embryo transfect ion studies with a TnI/beta-galactosidase reporter construct that demonstrated the existence of a functional TnI promoter within 1.5 kb of ATG-upstream DNA Moreover, RT-PCR experiments showed that TnI/beta-galactosidase transcripts contained the 16 nt mRNA common 5' end sequence despite the absence of this sequence from the transcriptional template construct. / This data is a demonstration of SL trans-splicing and represents the first reported case of SL trans-splicing among the deuterostomes. It suggests that SL trans-splicing may represent an ancestral metazoan feature. Moreover, because vertebrates probably arose from ascidian-like ancestors, it may be that many vertebrate genes, none of which are thought to undergo SL trans-splicing, are descended from trans-spliced ancestral genes. Such a history could explain the common occurrence in vertebrate genes of 5' untranslated region enhancer-containing introns.

A long-range research interest of this laboratory is the evolution of the transcriptional control mechanisms of the vertebrate troponin I (TnI) gene family. It is likely that the vertebrate TnI gene family arose from a single TnI gene present in early chordate ancestors. Analysis of transcriptional control mechanisms of the TnI gene of a primitive chordate, such as the ascidian Ciona intestinalis, may therefore provide insight into the regulatory mechanisms of the vertebrate ancestral TnI gene. As an initial step in such a study, I localized transcriptional control regions within 1.5 kb of 5'-flanking DNA of the Ciona TnI gene. I prepared a series of deletion constructs in which Ciona TnI 5'-flanking DNA segments were fused to a nuclear-targeted beta-galactosidase reporter gene. Constructs were introduced into fertilized Ciona eggs by electroporation, and following development up to the mid tailbud stage (12 h), reporter gene expression was assessed by whole-mount beta-galactosidase histochemistry. (Abstract shortened by UMI.)