Regulation of cardiac muscle contraction: effect of tropomyosin isoform expression and cardiomyopathy mutations in tropomyosin and troponin

Regulation of the acto-myosin interaction is mediated by the Ca2+ -induced changes in the properties of the thin filament proteins tropomyosin and troponin. Two major tropomyosin isoforms, a and 13, are expressed in striated muscle with larger amounts of 13 expressed in slow-contracting skeletal muscle and slower beating hearts of big mammals. The regulatory properties of a and 13 tropomyosin were defined in terms of the three-state model of regulation. The Ca2+ - dependent regulation of the thin filament was identical for a and 13 in the presence of skeletal troponin proteins. However, 13 tropomyosin-containing thin filaments were more Ca2+ sensitive in the presence of cardiac troponin. The thermal stability of the two isoforms was also examined using Differential Scanning Calorimetry. 13 tropomyosin was significantly less thermostable than a tropomyosin as it is partially unfolded at 37°C when free in solution. In the presence of actin, both u and 13 tropomyosin stability is increased. However, 13 tropomyosin unfolding starts at 40°C, a temperature the body can reach. These findings provide an explanation for the low level of 13 tropomyosin expression in the heart and the fact that it is predominantly found associated with a tropomyosin as a heterodimer. Familial Hypertrophy Cardiomyopathy (FHC) is an autosomal dominant disease, which causes sudden death of young and healthy individuals. The effect of FHC mutations expressed in tropomyosin and troponin proteins on the regulatory process was investigated. Two mutations in u tropomyosin (D175N and EI80G), and three mutations in trponin (G203S and K206Q in TnI, L29E in TnC) were examined. Both D175N and E180G increased the Ca2+ sensitivity of the system when associated with cardiac troponin Also, both mutations decreased the local stability of tropomyosin when free in solution In the presence of actin, D 17 5N tropomyosin thermal stability was indistinguishable from wild type. No detectable changes in the regulatory function of the thin filament were observed with troponin mutations G203S in troponin I and L29E in troponin C. However, K206Q mutation in troponin I caused an increase in the Ca2+ sensitivity interestingly only when troponin I was phosphorylated

Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells

Regulation of cardiac muscle contraction : effect of tropomyosin isoform expression and cardiomyopathy mutations in tropomyosin and troponin

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Role of tropomyosin isoforms in the calcium sensitivity of striated muscle thin filaments

We have expressed alpha & beta isoforms of mammalian striated muscle tropomyosin (Tm) and alpha-Tm carrying the D175N or E180G cardiomyopathy mutations. In each case the Tm carries an Ala-Ser N-terminal extension to mimic the acetylation of the native Tm. We show that these Ala-Ser modified proteins are good analogues of the native Tm in the assays used here. We go on to use an in vitro kinetic approach to define the assembly of actin filaments with the Tm isoforms with either a cardiac or a skeletal muscle troponin (cTn, skTn). With skTn the calcium sensitivity of the actin filament is the same for alpha & beta-Tm and there is little change with the mutant Tms. For cTn switching from alpha to beta-Tm causes an increase of calcium sensitivity of 0.2 pCa units. D175N is very similar to the wild type alpha-Tm and E180G shows a small increase in calcium sensitivity of about 0.1 pCa unit. The formation of the switched-off blocked-state of the actin filament is independent of the Tm isoform but does differ for cardiac versus skeletal Tn. The in vitro assays developed here provide a novel, simple and efficient method for assaying the behaviour of expressed thin filament proteins

The regulation of Myosin binding to actin filaments by lethocerus troponin

Lethocerus indirect flight muscle has two isoforms of troponin C, TnC-F1 and F2, which are unusual in having only a single C-terminal calcium binding site (site IV, isoform F1) or one C-terminal and one N-terminal site (sites IV and II, isoform F2). We show here that thin filaments assembled from rabbit actin and Lethocerus tropomyosin (Tm) and troponin (Tn) regulate the binding of rabbit myosin to rabbit actin in much the same way as the mammalian regulatory proteins. The removal of calcium reduces the rate constant for S1 binding to regulated actin about threefold, independent of which TmTn is used. This is consistent with calcium removal causing the TmTn to occupy the B or blocked state to about 70% of the total. The mid point pCa for the switch differed for TnC-F1 and F2 (pCa 6.9 and 6.0, respectively) consistent with the reported calcium affinities for the two TnCs. Equilibrium titration of S1 binding to regulated actin filaments confirms calcium regulated binding of S1 to actin and shows that in the absence of calcium the three actin filaments (TnC-F1, TnC-F2 and mammalian control) are almost indistinguishable in terms of occupancy of the B and C states of the filament. In the presence of calcium TnC-F2 is very similar to the control with approximately 80% of the filament in the C-state and 10-15% in the fully on M-State while TnC-F1 has almost 50% in each of the C and M states. This higher occupancy of the M-state for TnC-F1, which occurs above pCa 6.9, is consistent with this isoform being involved in the calcium activation of stretch activation. However, it leaves unanswered how a C-terminal calcium binding site of TnC can activate the thin filament

A regionally based precision medicine implementation initiative in North Africa:The PerMediNA consortium

International audiencePrecision Medicine is being increasingly used in the developed world to improve health care. While several Precision Medicine (PM) initiatives have been launched worldwide, their implementations have proven to be more challenging particularly in low- and middle-income countries. To address this issue, the “Personalized Medicine in North Africa” initiative (PerMediNA) was launched in three North African countries namely Tunisia, Algeria and Morocco. PerMediNA is coordinated by Institut Pasteur de Tunis together with the French Ministry for Europe and Foreign Affairs, with the support of Institut Pasteur in France. The project is carried out along with Institut Pasteur d’Algérie and Institut Pasteur du Maroc in collaboration with national and international leading institutions in the field of PM including Institut Gustave Roussy in Paris. PerMediNA aims to assess the readiness level of PM implementation in North Africa, to strengthen PM infrastructure, to provide workforce training, to generate genomic data on North African populations, to implement cost effective, affordable and sustainable genetic testing for cancer patients and to inform policy makers on how to translate research knowledge into health products and services. Gender equity and involvement of young scientists in this implementation process are other key goals of the PerMediNA project.In this paper, we are describing PerMediNA as the first PM implementation initiative in North Africa. Such initiatives contribute significantly in shortening existing health disparities and inequities between developed and developing countries and accelerate access to innovative treatments for global health