In 1980, the World Health Organization task force first sought to define and classify cardiomyopathies. They defined cardiomyopathies as "heart muscle diseases of unknown cause" with three main classifications including: hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy [1]. Over the next three decades it became patently obvious that this simple definition was not sufficient to describe the complex heterogeneity of diseases present in the patient population. More robust definitions were necessary for mechanistic links to be established and meaningful therapeutics to be developed. Since then the accepted definition of a cardiomyopathy has evolved and the classifications have greatly expanded. The most recent definition from the American Heart Association Council on Clinical Cardiology states: Cardiomyopathies are a heterogeneous group of diseases of the myocardium associated with mechanical and/or electrical dysfunction that usually (but not invariably) exhibit inappropriate ventricular hypertrophy or dilatation and are due to a variety of causes that frequently are genetic. Cardiomyopathies either are confined to the heart or are part of generalized systemic disorders, often leading to cardiovascular death or progressive heart failure–related disability [2]. This latest definition (2006) reflects the growing recognition of molecular genetics as a key factor in the development of cardiomyopathies and highlights the ever-growing complexity of disease classification. Today the genetic basis of HCM and DCM is widely recognized yet our understanding of the precise mechanisms underlying the disease remains unclear. To add to this disconnect, by the time patients become symptomatic, pathology has progressed past the initial phase, where meaningful treatment could occur, to advanced end-stage pathology. By this time often the only treatment options available become "blunt sword" therapeutics that are non-specific and used primarily for symptom management. In fact, over the last 3 decades there has been a marked decline in the innovation of cardiovascular pharmaceuticals owed partially to the vast complexity of disease presentation and progression [3]. In this dissertation, I will focus on a genetic sarcomeric DCM caused by a mutation in alpha-tropomyosin (Tm). Using novel accurate mouse models as a tool we will define the mechanism by which it leads to disease, investigate how disease severity due to the mutation is modified in an age-dependent manner, and examine what this mechanism could mean in the larger picture of cardiomyopathic disease progression. I hope to convince you that by using accurate models of this DCM at multiple levels of biological complexity to tease out the precise mechanisms of disease we can establish meaningful genotype-phenotype relationships that could lead to the development of specific novel therapeutics.

The progression of genetically inherited cardiomyopathies from an altered protein structure to the clinical presentation of the disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information of multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational methods to characterize the baseline function and structure of the Tm overlap, as well as the effects that mutations causing divergent patterns of ventricular remodeling have on both structure and function.

Diss. (sammanfattning) Uppsala : Univ., 2007. / Härtill 4 uppsatser.

No description available.

Seafood including crustaceans, on ingestion, are known to provoke gastrointestinal as well as systemic allergic reactions. Crustaceans are aquatic arthropods with a chitinous exoskeleton and include shrimp, lobster, prawn and crab. Earlier studies in our laboratory have led to the identification and characterization of three allergens from shrimp, designated as Sa-I, Sa-I1 and Sa-III. The former two were shown to be heat stable proteins with a mol. wt. of 8.4 and 34 kDa respectively, while Sa-III was identified as tRNA Arg and TRNATyr ). Sa-II was found to be the major allergen contributing to more than 50% of the allergenic activity. There are several reports on the existence of cross-reactivity among atopic allergens, in particular food allergens. It is well known that individuals with shrimp allergy often complain of adverse reactions following the ingestion of other re1ated crustaceans. Recognition of crustacea as a group causing adverse reactions in sensitive individuals has a basis in the close phylogenetic relationship of shrimp, lobster, crab and prawn. Thus, one could expect appreciable similarity in the IgE binding epitopes of the offending allergens from related crustaceans. The present study was, therefore, aimed towards the identification of the major cross-reacting crustacean allergen and localization of its IgE binding epitopes. Cross-reactivity among a1lergens from shrimp, prawn, crab and lobster was evaluated by immunochemical methods. Antigenic cross-reactivity was established by immunodiffusion using shrimp-specific rabbit IgG. Competitive ELlSA inhibition experiments using sera of shrimp sensitive patients revealed a high degree of allergenic cross-reactivity between different crustaceans. SDSPAGE and immunoblot analysis using the sera of shrimp sensitive patients have identified a 34 kDa protein as the cross-reacting crustacean allergen. Using shrimp as a model system and Sa-II as a representative crustacean allergen, further studies were carried out to get an insight into the structural and molecular basis of allergenic cross-reactivity. The strategies adopted were, (1) to raise allergen specific anti-idiotypic antibodies and explore the possibility of using these anti-idiotypic antibodies as surrogate allergens for diagnosis of crustacea allergy and (2) to identify the IgE binding epitopes on the major shrimp allergen Sa-II, which may be shared by the 34 kDa allergen from the related crustaceans. In order to explore idiotypic, anti-idiotypic and anti-anti-idiotypic responses to Sa-II, Balb/c mice were immunized with affinity purified human idiotypic antibodies directed against the purified allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated using rabbit idiotypic antibodies raised against the same allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp sensitive patients. Immunization of Balb/ c mice with affinity purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. The induction of anti-anti-idiotypic antibodies functionally identical to allergen-specific idiotypic antibodies confirmed that the anti-idiotypic antibodies generated, are indeed a mirror image of the allergen. The present study thus provides evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens. These anti-anti-idiotypic antibodies not only recognized Sa-II, but also the 34 kDa allergen from prawn, lobster and crab. Cross-reactivity studies using polyclonal sera of shrimp sensitive patients and Sa-II anti-anti-idiotypic antibodies have attributed the allergenic cross-reactivity observed among the related crustaceans to the presence of highly conserved IgE binding epitopes on the 34 kDa crossreacting allergen from shrimp, crab, lobster and prawn. In order to identify the igE binding epitopes on Sa-11, it was subjected to limited tryptic digestion and the peptides were separated by reverse phase HPLC. Amino acid sequence analysis of these peptides and several other peptides generated by Asp N and Lys C treatment revealed an 861 homology with the muscle protein tropomyosin from the fruit fly Drosophila melanogaster, suggesting that the major shrimp allergen is tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from shrimp and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8-5.4. In the presence of 6 M urea, the mobility of both Sa-I1 and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp sensitive patients as assessed by competitive ELISA inhibition and immunoblot analysis. Tropomyosin, similar to Sa-I1 was subjected to limited tryptic digestion and the tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were found to be super imposable. Dot blot immunoassay and competitive ELISA inhibition assay using the sera of shrimp sensitive patients identified two peptides, 6 and 9 that exhibited allergenic activity. Both the peptides were purified to homogeneity and sequenced. Peptide 6 is a nonapeptide corresponding to the amino adds 153-161 and peptide 9 has 17 amino acids corresponding to the aminoacid residues 50-66. The peptides individually blocked upto 50% the binding of allergen-specific IgE to hropomyosin. Sa-II specific mouse anti-anti-idiotypic antibodies recognized not only tropomyosin, but also the two allergenic peptides, thus confirming that these peptides represent the major IgE binding epitopes. The IgG binding activity was found to be associated with peptides 6 and 9 as assessed by dot blot immunoassay using the sera of shrimp sensitive patients. Thus, it was found that both IgG and IgE binding epitopes on shrimp tropomyosin are identical. Tropomyosins from both phylogenetically related and unrelated species were assessed for allergenic activity using the sera of shrimp sensitive patients. It was found that allergenic activity was associated with tropomyosins from related crustaceans and from Drosophila melanogaster which shares 86% homology with shrimp tropornyosin. However, tropomyosins from totally unrelated species like yeast, chicken, bovine, rat, rabbit and human did not exhibit allergenic activity. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes with the corresponding regions of bopomyosins from different species confirmed lack of allergenic cross-reactivity. The allergenic peptides 6 and 9 were able to inhibit the binding of tropomyosins from related crustaceans to shrimp tropomyosin-specific IgE antibodies to the same extent, confirming the presence of highly conserved IgE binding epitopes. It has been established for the first time that the major crustacean allergen is the heat stable muscle protein, tropomyosin, and extensive cross-reactivity between different members of crustacea is due to the presence of highly conserved IgE binding epitopes on tropomyosins from these sources. Thus, from the present study, information with respect to the amino acid sequence of tropomyosin and localization of its 1gE binding epitopes, could be used to design synthetic peptides corresponding to the B cell and T cell epitopes which would find application in the diagnosis and desensitization of individuals allergic to crustacea.

Skeletal muscle fatigue is defined as a loss in the force/velocity generating capacity of a muscle. A portion of the loss in function is attributable to effects of acidosis (i.e. low pH) on the regulatory proteins, troponin and tropomyosin (Tm), which regulate the binding of myosin and actin in a calcium (Ca++) dependent manner. However, the relative role of troponin and Tm on myosin-actin function during acidosis is not clear, nor are the mechanisms underlying these effects. PURPOSE: To determine the role of Tm in the acidosis-induced depression of muscle function using isolated muscle proteins in an in vitro motility assay. METHODS: Three mutant constructs of Tm were expressed by replacing the two amino acid (histidine) residues most likely affected by low pH with alanine residues (H153A, H276A, H153A/H276A). These mutant constructs were compared to wild-type Tm (wt-Tm) in order to test whether the acidosis-induced charge change of the histidine amino acid governs the pH-dependent alteration of tropomyosin and therefore the decrease in maximal RTF velocity and Ca++-sensitivity. The effect of acidosis on regulated thin filament (RTF) function was determined by assessing the impact of low pH (pH 6.8) versus neutral pH (pH 7.4) on myosin’s ability to move RTFs in the motility assay as a function of increasing levels of Ca++. This was done separately for the wt-Tm and each structural variant. RESULTS: A two-way ANOVA (pH x Tm construct) revealed that acidosis significantly (p<0.05) depressed the maximal sliding velocity of the RTFs across all versions of Tm, but that the magnitude of the depression was similar among the wt and all of the Tm mutants. Acidosis did not significantly depress the sensitivity to Ca++ under the unloaded conditions of this assay (p>0.05). CONCLUSIONS: These data suggest that the histidine residues in tropomyosin do not mediate the acidosis-induced depression in contraction velocity observed during muscle fatigue. However, since these residues may be more important in mediating the depression of force, we are currently testing the impact of the three mutant Tm constructs on the acidosis-induced depression in Ca++-sensitivity using a loaded in vitro motility assay.

No description available.

Diseases of heart muscle, such as hypertrophic and dilated cardiomyopathy, are often caused by mutations in proteins of the sarcomere, including actin, troponin, tropomyosin, and myosin. The molecular mechanisms of disease-causing mutations remain unclear because the process of cardiac muscle contraction and the corresponding mutational insults are incompletely defined. To elucidate the underlying mechanisms of cardiac muscle contraction, its regulation, and the effects of disease-causing mutations, the structures of sarcomeric protein assemblies must first be solved. In this dissertation we use interdisciplinary structural biology techniques, including cryo-electron microscopy (cryo-EM), protein-protein docking, and molecular dynamics simulations to investigate the interactions between actin, tropomyosin, and myosin. This structural work is foundational in identifying the molecular effects of mutations. In the first project, we present a novel cryo-EM structure of the cardiac-isoform actomyosin-tropomyosin complex. This structure, which utilizes bovine masseter β-myosin, provides the foundation for understanding the molecular effects of cardiomyopathy-causing mutations that occur at the actomyosin interface. Furthermore, by pairing our structure with protein-protein docking methods and molecular dynamics simulations, we identify complementary and periodic electrostatic interactions between the myosin surface loop 4 and tropomyosin. We hypothesize that these interactions are essential in switching between contraction- and relaxation-mediating states. In a follow up study, we test our myosin loop 4 hypotheses by creating a human cardiac β-myosin all-glycine loop 4 mutant, which abolishes nearly all electrostatic interactions between myosin and tropomyosin. After designing the mutant, we solve the cryo-EM structures of the wild-type and mutant actin-myosin-tropomyosin complexes to high resolution. Our structures confirm that the loop 4 mutant abolishes its interaction with tropomyosin and suggests that the tropomyosin cable on mutant actomyosin filaments is shifted to a new position. Subsequent molecular dynamics simulations corroborate our cryo-EM finding that tropomyosin on mutant actomyosin is displaced from the wild type position. Interaction energy calculations derived from these simulations suggest that the mutant position is significantly less stable than the wild-type. This work provides further evidence that loop 4 interactions are key in stabilizing tropomyosin position during contraction. Finally, to extend our work on the human cardiac actomyosin-tropomyosin complex, we provide insights into the ADP release step of the cardiac β-myosin kinetic cycle. Here, we use a composite method of helical and single-particle cryo-EM reconstruction techniques to solve the structures of the human cardiac actin-myosin-tropomyosin filament in the presence and absence of ADP-Mg2+. This work elucidates the structural basis of cardiac β-myosin ADP release and provides insight into the force-sensing mechanism of the cardiac motor. Lastly, we use our structures to probe how cardiomyopathy-causing mutations potentially disrupt the ADP-to-rigor transition, leading to altered myosin contractility. Overall, the structures solved in this dissertation generate fundamental understanding about the function of the cardiac thin filament and the motor protein, myosin. Moreover, this research provides a framework that connects the initial molecular insults of mutations to the disruption of proper regulation that leads to pathological progression. / 2023-08-24T00:00:00Z

Cardiac contraction at the level of the sarcomere is regulated by the thin filament (TF) composed of actin, alpha tropomyosin (TPM), and the troponin (Tn) complex (cTnT: cTnC: cTnI). The "gate-keeper" protein, α-TPM, is a highly conserved α-helical, coiled-coil dimer that spans actin and regulates myosin-actin interactions. The N-terminus of one α-TPM dimer inter-digitates with the C-terminus of the adjacent dimer in a head-to-tail fashion forming the flexible and cooperative TPM-overlap that is necessary for myofilament activation. Two dilated cardiomyopathy (DCM) causing mutations in TPM (D84N and D230N) and one hypertrophic cardiomyopathy (HCM) causing mutation (E62Q), all identified in large, unrelated, multigenerational families, were utilized to study how primary alterations in protein structure cause functional deficits. We hypothesize that structural changes from a single point mutation propagate along the -helical coiled-coil of TPM, thus affecting its regulatory function. Structural effects of the mutations studied via differential scanning calorimetry (DSC) on TPM alone revealed significant changes in the thermal unfolding temperatures of both the C- and N-termini for all mutants compared to WT, indicating that mutational effects propagate to both ends of TPM, thus affecting the overlap region. Although, of note, the proximal termini to the mutation has shown more significant structural changes compared to WT. DSC analysis on fully reconstituted TF’s (Tn:TPM:Actin) revealed effects on the TPM-Actin cooperativity of activation, affecting interaction strength (thermal stability), and the rigidity of TPM moving along actin (FWHM). To characterize the resultant functional effect of these discrete changes in thermal stability and TPM rigidity, ATPase assays were used to measure actomyosin activation in the presence and absence of Ca2+. Together, these data will provide a molecular level understanding of the structural and functional deficits caused by these mutations to help elucidate the mechanisms leading to disease.

Cell motility is a fundamental process, enabling cells to migrate, for instance during embryogenesis, tissue repair and defense. Force is generated by two protein systems, which also participate in cell proliferation, control macromolecular and organelle distribution and determine the fine structure of the cell interior. The major components of these are actin and tubulin, respectively, and they are referred to as the microfilament and the microtubule systems. This thesis focuses on tropomyosin, one of many microfilament associated proteins coupled to actin dynamics and organization and expressed in several isoform variants. Altered distribution and isoform expression of tropomyosin are signatures of malignant cells and are dealt with in the current thesis. The presence of tropomyosin isoforms in protruding lamellipodia of migrating cells is demonstrated, and a method to fractionate tropomyosin depending on its organization in an easily extractable, and a more tightly bound cytoplasmic form is presented. Analysis of the loosely associated tropomyosin fraction by gel filtration chromatography revealed that most of the tropomyosins in this fraction exist in a multimeric form. It was also observed that the distribution of tropomyosin varied between non-transformed and transformed cells with most of the isoforms enriched in the loosely bound fraction in the latter category of cells. Possibly this reflects the extensive reorganization of the microfilament system observed in cancer cells and which, depending on the context, can be normalized by introduction of certain tropomyosin isoforms. Many anti-cancer drugs target the microtubule system, inhibit cell division and promote apoptosis. Here it is shown that picropodophyllin, which has promising anticancer properties has a destabilizing effect on microtubules and via the microfilament system causes cells to detach from their substratum. Furthermore, picropodophyllin interferes with stimulation of the insulin-like growth factor receptor, which is involved in growth stimulation, differentiation and survival and whose expression is up-regulated in cancer cells.