Regulation of G protein-coupled receptor kinase subtypes by calcium sensor proteins

AbstractG protein-coupled receptor homologous desensitization is intrinsically related to the function of a class of S/T kinases named G protein-coupled receptor kinases (GRK). The GRK family is composed of six cloned members, named GRK1 to 6. Studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor-specific CSP recoverin through direct binding. Several other recoverin homologues (including NCS 1, VILIP 1 and hippocalcin) are also able to inhibit GRK1. The ubiquitous calcium-binding protein calmodulin (CaM) can inhibit GRK5 with a high affinity (IC50=40–50 nM). A direct interaction between GRK5 and Ca2+/CaM was documented and this binding does not influence the catalytic activity of the kinase, but rather reduced GRK5 binding to the membrane. These studies suggest that CSP act as functional analogues in mediating the regulation of different GRK subtypes by Ca2+. This mechanism is, however, highly selective with respect to the GRK subtypes: while GRK1, but not GRK2 and GRK5, is regulated by recoverin and other NCS, GRK4, 5 and 6, that belong to the GRK4 subfamily, are potently inhibited by CaM, which had little or no effect on members of other GRK subfamilies

Platinated Nucleotides are Substrates for the Human Mitochondrial Deoxynucleotide Carrier (DNC) and DNA Polymerase g: Relevance for the Development of New Platinum-Based Drugs.

cis-[PtCl2(NH3)2] (cisplatin) is among the highest effective antitumor drugs used for the chemotherapeutic treatment of a broad range of malignancies. Recently, alongside with the classical direct bond to DNA, an alternative mechanism of action mediated by N7 platinated nucleotides has been suggested for cisplatin. Considering that mitochondria play an important role in cell death activation and in a significant portion of the clinical activity and pharmacological properties associated with cisplatin, aim of this research was to evaluate the possibility that platinated deoxynucleotides, as the model complex [Pt(dien)(N7-5’-dGTP)] (1), dien=diethylenetriamine, could be transported into mitochondria and then incorporated into mtDNA. The kinetic characterization has revealed that the mitochondrial deoxynucleotide carrier (DNC) transports complex 1 with high affinity. Finally, a highly efficient in organello DNA synthesis system, followed by ICP-AES, has demonstrated that [Pt(dien)(N7-5’-dGTP)] is incorporated in the mitochondrial DNA by DNA polymerase g. These results may have critical implications in the development of new generations of anticancer and/or antiviral nucleotide analogues with more specific cellular targets and fewer side effects

Role of G Protein-coupled Receptor Kinase 4 and β-Arrestin 1 in Agonist-stimulated Metabotropic Glutamate Receptor 1 Internalization and Activation of Mitogen-activated Protein Kinases

The metabotropic glutamate 1 (mGlu(1)) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu(1) receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu(1) receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Galpha(q). In cells transfected with GRK4 and exposed to agonist, beta-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu(1) receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of beta-arrestin V53D dominant negative mutant, which did not affect the mGlu(1) receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu(1) receptor. The agonist-stimulated differential sorting of the mGlu(1) receptor and beta-arrestin as well as the activation of MAP kinases by mGlu(1) agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of beta-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing beta-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu(1) receptor by different mechanisms and that beta-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule

Efficacy Evaluation of Cu- and Ag-Based Antibacterial Treatments on Polypropylene Fabric and Comparison with Commercial Products

Filter masks are disposable devices intended to be worn in order to reduce exposure to potentially harmful foreign agents of 0.1–10.0 microns. However, to perform their function correctly, these devices should be replaced after a few hours of use. Because of this, billions of non-biodegradable face masks are globally discarded every month (3 million/minute). The frequent renewal of masks, together with the strong environmental impact of non-biodegradable plastic-based mask materials, highlights the need to find a solution to this emerging ecological problem. One way to reduce the environmental impact of masks, decrease their turnover, and, at the same time, increase their safety level is to make them able to inhibit pathogen proliferation and vitality by adding antibacterial materials such as silver, copper, zinc, and graphene. Among these, silver and copper are the most widely used. In this study, with the aim of improving commercial devices’ efficacy and eco-sustainability, Ag-based and Cu-based antibacterial treatments were performed and characterized from morphological, compositional, chemical–physical, and microbiological points of view over time and compared with the antibacterial treatments of selected commercial products. The results demonstrated the good distribution of silver and copper particles onto the surface of the masks, along with almost 100% antibacterial capabilities of the coatings against both Gram-positive and Gram-negative bacteria, which were still confirmed even after several washing cycles, thus indicating the good potential of the developed prototypes for mask application

Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster

none13noCoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.Vozza, Angelo; Leonardis, Francesco De; Paradies, Eleonora; Grassi, Anna De; Pierri, Ciro Leonardo; Parisi, Giovanni; Marobbio, Carlo Marya Thomas; Lasorsa, Francesco Massimo; Muto, Luigina; Capobianco, Loredana; Dolce, Vincenza; Raho, Susanna; Fiermonte, GiuseppeVozza, Angelo; Leonardis, Francesco De; Paradies, Eleonora; Grassi, Anna De; Pierri, Ciro Leonardo; Parisi, Giovanni; Marobbio, Carlo Marya Thomas; Lasorsa, Francesco Massimo; Muto, Luigina; Capobianco, Loredana; Dolce, Vincenza; Raho, Susanna; Fiermonte, Giusepp

How Detergent Impacts Membrane Proteins: Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine.

Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent-membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states

DMTs and Covid-19 severity in MS: a pooled analysis from Italy and France

We evaluated the effect of DMTs on Covid-19 severity in patients with MS, with a pooled-analysis of two large cohorts from Italy and France. The association of baseline characteristics and DMTs with Covid-19 severity was assessed by multivariate ordinal-logistic models and pooled by a fixed-effect meta-analysis. 1066 patients with MS from Italy and 721 from France were included. In the multivariate model, anti-CD20 therapies were significantly associated (OR = 2.05, 95%CI = 1.39–3.02, p < 0.001) with Covid-19 severity, whereas interferon indicated a decreased risk (OR = 0.42, 95%CI = 0.18–0.99, p = 0.047). This pooled-analysis confirms an increased risk of severe Covid-19 in patients on anti-CD20 therapies and supports the protective role of interferon