Fatigue and skeletal muscle weakness are problems associated with congestive heart failure. Research does not support the theory that the affected cardiac function is responsible for the fatigue. During skeletal muscle fatigue, calcium handling is altered. Thus, the fatigue associated with congestive heart failure could be attributed to altered calcium handling. The main proteins involved in calcium release are the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The main proteins involved in calcium uptake are the fast and slow isoforms of sarco(endo)plasmic reticulum calcium ATPase (SERCA 1 and SERCA 2 respectively). Calsequestrin (Csq) and calmodulin (CaM) play regulatory roles in calcium handling. Changes in the levels of these proteins could explain alterations in calcium handling and subsequent muscle function. The purpose of this study was to use a genetic model of heart failure, the SHHF rat, to examine the levels of regulatory calcium handling proteins to determine if changes in the amounts of RyR, DHPR, SERCA1, SERCA2, Csq and CaM are altered in congestive heart failure. A significant decrease was found in the amounts of RyR, DHPR, and SERCA 1 of the SHHF gastrocnemius and diaphragm samples in comparison to the control. There was no significant difference found in the amounts of CaM or SERCA 2 between the two groups. Csq was not found to be statistically different between the two groups of the gastrocnemius samples. However, there was an increase in Csq in the SHHF diaphragm samples in comparison to the control. In conclusion, the calcium handling proteins are affected in the genetic model of heart failure. These changes could explain previous reports of altered calcium handling within the skeletal muscles of congestive heart failure animals. / Master of Science

No description available.

No description available.

Discrete changes in the amplitude, frequency, and cellular localisation of calcium ion (Ca2+) transients encode information about numerous stimuli and function to mediate stimulus-specific responses. Cytoplasmic Ca2+ (Ca2+cyt) undergoes circadian oscillations in concentration that appear to be under the control of the same endogenous oscillator that regulates expression of genes in the photoperiodic-flowering pathway. It is currently not known whether these circadian [Ca2+cyt] oscillations are biochemical artefacts or are decoded and function to transduce clock dependent responses. Calmodulin (CaM) is a primary node in Ca2+ signalling in plants and as such is a promising target for investigating the role of Ca2+ in clock-controlled processes. In this study, Arabidopsis thaliana were treated with experimentally validated concentrations of pharmacological CaM inhibitors. Under inductive photoperiods (16 h light : 8 h dark), CaM inhibition was found to increase developmental flowering time, whilst under non-inductive photoperiods no such changes were evident. Inhibition of CaM led to changes in expression of the key clock gene TIMING OF CAB EXPRESSION 1 and flowering time genes, CONSTANS and FLOWERING LOCUS T and removed repression of flowering in darkness. These observations are consistent with CaM modulating the activity of the putative clock component GIGANTEA and the proteasomal targeting protein SUPPRESSOR OF PHYA-105. Due to the unwanted side effects often generated by chemical CaM inhibitors, a peptide inhibitor of CaM comprising a green fluorescent protein / calspermin fusion and labelled smGN was developed. Surface plasmon resonance analysis and affinity chromatography showed smGN to have extremely high selectivity for, and affinity to, CaM and to function as a powerful inhibitor of CaM in vitro. Further work on the methodology used to deploy smGN as a recombinant alternative to chemical CaM inhibitors in planta is also described.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Surfactant deficiency leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. These are features of the acute lung injury syndrome, characterized by a strong pro-inflammatory component where cytokines or bacteria infections greatly impair surfactant DPPtdCho biosynthesis. The key enzyme needed to produce surfactant DPPtdCho is a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCTalpha). Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilizes CCTalpha from actions of the proteinase, calpain. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Q243 not only resulted in loss of CaM binding, but also led to complete calpain resistance in vitro and in vivo. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under pro-inflammatory stress. We further show that CCTalpha does not undergo polyubiquitination and proteasomal degradation. Rather, the enzyme is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS resulting in disruption of its interaction with importin, nuclear exclusion, and subsequent degradation within the lysosome. Importantly, by using CCTalpha-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTalpha monoubiquitination masks its NLS resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking of a critical regulatory enzyme in vivo. Last, we identify FBXL2 as a novel F-box E3 ubiquitin ligase that targets CCTalpha for degradation. Interestingly, FBXL2 also interacts with CaM, and CaM directly disrupts CCTalpha and FBXL2 interaction. This study demonstrates in the first time that adenoviral gene transfer of CaM attenuates the deleterious effects of P. aeruginosa infection by improving several parameters of pulmonary mechanics in animal models of sepsis-induced acute pulmonary injury. Collectively, these studies reveal a novel regulatory mechanism for phosphatidylcholine synthesis that may provide important clues to understanding the pathobiology of acute lung injury.

The cardiac ryanodine receptor (RyR2) plays a key role in excitation-contraction coupling (ECC). Mutations in RyR2 are known to be linked to the arrhythmogenic disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT), a deadly disease which is characterized by a leak of calcium from sarcoplasmic reticulum and a decrease in calmodulin (CaM) binding. A novel drug, 84F2, shown to inhibit arrhythmias in RyR2-R176Q heterozygous CPVT mouse hearts (2.5 µg/kg), decrease spark frequency in cells derived from CPVT mice (IC50 = 35 nM), and inhibit RyR2 single channel activity at low nanomolar concentrations (IC50 = 8 nM). When CaM is added back to RyR2, 84F2's ability to inhibit channel activity is suppressed approximately 250 fold. A metabolite of 84F2, 78F3, is shown to also be active in the inhibition of RyR2. We propose that 84F2 decreases arrhythmias by binding to the CaM deficient RyR2, but does not affect normal ECC when CaM is present. This work characterizes for the first time a class of drugs whose inhibitory affects are dependent upon the removal of CaM from RyR2.

Calcineurin (CaN), a heterodimeric Ca2+-calmodulin-dependent Ser/Thr phosphatase, regulates diverse pathways, from stress responses in yeast to T-cell activation and cardiac hypertrophy in humans. Calmodulin (CaM), an essential mediator of calcium–dependent signaling pathways, activates CaN in the presence of calcium by binding to an intrinsically disordered region of the enzyme and altering its conformation. My hydrodynamic studies have determined that CaM participates in a 1:1 complex with the CaM-binding domain of βCaN (CaNp, residues 400–423). To explore the molecular mechanism of CaM association with CaN, I have used spectroscopic methods to determine the calcium-dependent and domain–specific interactions of CaM with CaNp. These studies revealed that the affinity of CaM1–148 for CaNp was weak in the absence of calcium, and very high (Kd in the nM to pM range) in the presence of calcium. I have demonstrated that CaNp binding to CaM increases the calcium–binding affinity of each domain of CaM1–148 to a similar degree, thereby retaining the property of sequential calcium binding to the domains, with preference for sites in the C–domain. This allows the N–domain to lag in response to an increase in cellular calcium and perhaps contribute to the regulation of CaN in a manner distinct from that of the C–domain. NMR studies of calcium–saturated CaM1–148 demonstrated that the N–domain of CaM experienced a larger structural perturbation than the C–domain upon binding CaNp. Additional NMR studies revealed that CaNp adopts an anti–parallel orientation when bound to CaM, with the sole aromatic residue of CaNp contacting the N–domain of CaM. This contrasts with many CaM-target complexes in which the sole aromatic residue contacts the C–domain of CaM. Rigorous thermodynamic studies explored how mutations in the calcium-binding sites of mammalian CaM (mCaM) and mutations known to cause disruption of CaM–mediated ion channel regulation in Paramecia (PCaM) affected the allosteric interactions of the domains of CaM in the presence of CaNp. These studies demonstrated separable roles of the domains of CaM in recognition of CaNp. The consequences of a mutation depended upon its location within the complex. Collectively, research presented in this thesis provides insight into the mechanisms whereby the two domains of CaM contribute to recognition of CaN.

Graduation date: 2006

Essential metals like Ca2+ and Zn2+ play critical roles in biological processes through protein interactions. Conversely, non-essential metals (e.g., Gd3+ and Pb2+) also interact with proteins, often with toxic effects. Molecular metal toxicity is assumed to be due to ionic displacement, and studies have demonstrated that Pb2+ replaces Zn2+, Ca2+ and other essential metals in proteins. The focus of this work was to compare protein Ca2+ and Pb2+ -binding sites and to investigate a mechanism of Pb2+ toxicity in Ca2+-binding proteins, particularly the intracellular trigger protein calmodulin (CaM) which binds four Ca2+ ions and interacts with numerous molecular targets via Ca2+-induced conformational change. A statistical analysis of PDB structural data for Pb2+ and Ca2+-binding (EF-hand and non-EF-hand) proteins revealed fewer binding ligands in Pb2+ sites (4 ± 2), than non-EF-Hand (6 ± 2) and EF-Hand (7 ± 1) Ca2+-binding sites. Pb2+ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) sites. Interestingly, analyses of proteins where Pb2+ replaces Ca2+ (calmodulin) or Zn2+ (5-aminolaevulinic acid dehydratase) revealed structural changes presumably unrelated to ionic displacement. These results suggested that Pb2+ adopts diverse binding geometries and that opportunistic binding outside of known Ca2+-binding sites may play a role in molecular metal toxicity. Ca2+-binding affinities (Kd) using phenylalanine and tyrosine fluorescence were found to be 1.15 ± 0.68 X 10-5 M and 2.04 ± 0.02 X 10-6 M for the N- and C-terminal domains, respectively. The Kd for Pb2+-binding in the N-terminal domain, 1.40 ± 0.30 X 10-6 M, was 8-fold higher than Ca2+. Binding of Pb2+ in the C-terminal domain produced a biphasic response with Kd values 7.34 ± 0.95 X 10-7 M and 1.93 ± 0.32 X 10-6 M, suggesting a single higher affinity Pb2+-binding site in the C-terminal domain with nearly equivalent affinity for the remaining sites. Competitive effects of Pb2+ added to Ca2+-loaded CaM were examined using multiple NMR techniques. Pb2+ was found to displace Ca2+ only in the N-terminal domain, however structural/dynamic changes were observed in the central helix apparently due to Pb2+-binding in secondary sites. These data supported our hypothesis that CaM structure and function is altered by opportunistic Pb2+-binding.

Thesis (M. Phil.)--University of Hong Kong, 1984.