Effect of Cavβ Subunits on Structural Organization of Cav1.2 Calcium Channels

Background

Voltage-gated Ca_v1.2 calcium channels play a crucial role in Ca²⁺ signaling. The pore-forming α_1C subunit is regulated by accessory Ca_vβ subunits, cytoplasmic proteins of various size encoded by four different genes (Ca_vβ₁ - β₄) and expressed in a tissue-specific manner.

Methods and Results

Here we investigated the effect of three major Ca_vβ types, β_1b, β_2d and β₃, on the structure of Ca_v1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Ca_v1.2 to form clusters depends on the type of the Ca_vβ subunit present. The highest density of Ca_v1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac β_1b present. Ca_v1.2 channels containing β₃, the predominant Ca_vβ subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between α_1C and Ca_vβ in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Ca_v1.2 channels in the plasma membrane depends on the Ca_vβ type. The presence of different Ca_vβ subunits does not result in significant differences in the intramolecular distance between the termini of α_1C, but significantly affects the distance between the termini of neighbor α_1C subunits, which varies from 67 Å with β_1b to 79 Å with β₃.

Conclusions

Thus, our results show that the structural organization of Ca_v1.2 channels in the plasma membrane depends on the type of Ca_vβ subunits present.     

Préparations magistrales en radiopharmacie : contraintes liées à la mise en place

Use of radiopharmaceuticals in French nuclear medicine departments depends on marketing authorization and their development may be compromised by a limited return on investment. As an alternative, radiopharmaceuticals may also be prepared in the form of a magistral preparation, like in some European countries. In this case, these preparations are subjected to restrictions and requirements for radiopharmacies relating to quality assurance, facilities and equipment, quality of starting materials and final radiopharmaceutical products defined in French good preparation practice. Labelled tracers used as magistral preparations have to be prepared under the full responsibility of a radiopharmacist and used under the responsibility of the prescribing physician. Conditions of sufficient guarantees for the safety of the patient and adherence to pharmaceutical rules must be evaluated individually. However, this form of preparation intends to supply specific medical needs for an individual patient and is not an answer in the framework of development of radiopharmaceuticals.     

Genetic engineering of self-assembled protein hydrogel based on elastin-like sequences with metal binding functionality

Recombinant DNA methods have been exploited to enable the creation of protein-based block copolymers with programmable sequences, desired properties, and predictable three-dimensional structures. These advantages over conventional polymer counterparts facilitate the utility of this new class of biomaterials in a wide range of applications. In this project, we exploited the environmental application of protein-based block copolymers based on elastin-like protein (ELP) sequences. Triblock copolymers containing charged and hydrophobic segments were synthesized. Chain lengths of each segment were manipulated in order to maintain a gelation point below room temperature. Polyhistidine sequences were successfully incorporated into the hydrophilic segment without disruption of the self-assembled hydrogel formation. The microscopic structure was further investigated using laser confocal microscopy. The metal binding capability and capacity of resulting hydrogel were studied to demonstrate the functionality of polyhistidine and its environmental application for heavy metal removal. Reversibility of metal binding was demonstrated, indicating the cost-effectiveness of this hydrogel. Significantly, we envision that this versatile strategy of incorporating functional groups within a 3-D protein network provides new possibilities in creation of biomaterials with great control over structure-property relationships.     

Two mutations impair the stability and function of ubiquitin-activating enzyme (E1)

Protein ubiquitination plays critical roles in the regulation of multiple cellular processes including cell proliferation, signal transduction, oncogenesis, and hypoxic response. TS20 is a Balb3T3-derived cell line in which ubiquitination is inhibited by restrictive temperature. While TS20 has been used to elucidate the degradation of many important proteins including p53, p27, HIF-1α, and ornithine decarboxylase, the molecular basis of its temperature sensitivity has not been fully determined. We cloned full-length E1 cDNA from TS20. Sequencing analysis revealed two point mutations (nt736G to A and nt2313G to C) that lead to substitution of aa189A to T and aa714W to C, respectively. Transient transfection assays revealed that mutant E1 was less stable than its wild-type counterpart, and restrictive temperature (39°C) accelerated its degradation. Under permissive temperature, reverting aa714C to W significantly improved E1 stability and activity. Under restrictive temperature, reverting of both substitutions was required to fully restore E1 stability. Similar results were observed when the mutants were expressed in non-TS20 cells, indicating the mutations are sufficient for its temperature sensitive degradation observed in TS20 cells. Functionally, reverting aa714C to W was sufficient to facilitate the monoubiquitination of H2A and to support TS20 growth at 39°C. It also significantly improved the ubiquitination-dependent disposal of HIF-1α. Our data conclusively demonstrate that mutations introgenic to UVBE1 cause E1 instability, which leads to deficiency of E1 function. Our data establish the molecular basis for unambiguous interpretation of experimental data based on TS20 cells, and provide new insight into the structural determinants of E1 stability.     

Enantioselective conjugate addition of ketones to nitroalkenes catalyzed by pyrrolidine-sulfamides

A series of chiral pyrrolidine-sulfamides were prepared and examined as the catalysts for conjugate addition of ketones to nitroalkenes. Benzoic acid was identified as the most efficient additives for the transformation. Excellent enantioselectivities, diastereoselectivities, and yields were achieved for the reaction of cyclohexanone with β-aryl nitroethylenes under solvent free conditions. β-Isopropyl nitroethylene is also applicable and the product could be obtained with excellent enantioselectivity after extended reaction time. A comparison of the catalytic behaviors of pyrrolidine-sulfamide organocatalysts with different side chains demonstrates that the enantioselectivity is mainly controlled by the chiral pyrrolidine unit and the additional chiral center at the side chain exerts neglectable effects. The H-bonding interaction between the sulfamide and the nitro group is proposed to be crucial for the activation of the nitroalkene and the constitution of well-organized transition state.