1,721,103 research outputs found
Structural determinants of ligands recognition by the human mitochondrial basic amino acids transporter SLC25A29. Insights from molecular dynamics simulations of the c-state
Full text linkIn mitochondria, metabolic processes require the trafficking of solutes and organic molecules, such as amino acids. This task is accomplished by the Mitochondrial Carrier Family members (also known as SLC25), among which the SLC25A29 is responsible for the translocation of basic amino acids. In this regard, nitric oxide levels originated by the arginine mitochondrial catabolism have been shown to strongly affect cancer cells’ metabolic status. Furthermore, the metabolic disease saccharopinuria has been linked to a mitochondrial dysregulation caused by a toxic intermediate of the lysine catabolism. In both cases, a reduction of the activity of SLC25A29 has been shown to ameliorate these pathological conditions. However, no detailed structural data are available on SLC25A29. In the present work, molecular modelling, docking and dynamics simulations have been employed to analyse the structural determinants of ligands recognition by SLC25A29 in the c-state. Results confirm and reinforce earlier predictions that Asn73, Arg160 and Glu161, and Arg257 represent the ligand contact points I, II, and III, respectively, and that Arg160, Trp204 and Arg257 form a stable interaction, likely critical for ligand binding and translocation. These results are discussed in view of the experimental data available for SLC25A29 and other homologous carriers of the same family
Computational studies of the mitochondrial carrier family SLC25. Present status and future perspectives
Full text linkThe members of the mitochondrial carrier family, also known as solute carrier family 25 (SLC25), are transmembrane proteins involved in the translocation of a plethora of small molecules between the mitochondrial intermembrane space and the matrix. These transporters are characterized by three homologous domains structure and a transport mechanism that involves the transition between different conformations. Mutations in regions critical for these transporters' function often cause several diseases, given the crucial role of these proteins in the mitochondrial homeostasis. Experimental studies can be problematic in the case of membrane proteins, in particular concerning the characterization of the structure-function relationships. For this reason, computational methods are often applied in order to develop new hypotheses or to support/explain experimental evidence. Here the computational analyses carried out on the SLC25 members are reviewed, describing the main techniques used and the outcome in terms of improved knowledge of the transport mechanism. Potential future applications on this protein family of more recent and advanced in silico methods are also suggested
In silico analysis of huntingtin homologs in lower eukaryotes
Full text linkHuntington’s disease is a rare neurodegenerative and autosomal dominant disorder. HD is caused by a mutation in the gene coding for huntingtin (Htt). The result is the production of a mutant Htt with an abnormally long polyglutamine repeat that leads to pathological Htt aggregates. Although the structure of human Htt has been determined, albeit at low resolution, its functions and how they are performed are largely unknown. Moreover, there is little information on the structure and function of Htt in other organisms. The comparison of Htt homologs can help to under-stand if there is a functional conservation of domains in the evolution of Htt in eukaryotes. In this work, through a computational approach, Htt homologs from lower eukaryotes have been analysed, identifying ordered domains and modelling their structure. Based on the structural models, a putative function for most of the domains has been predicted. A putative C. elegans Htt-like protein has also been analysed following the same approach. The results obtained support the notion that this protein is a orthologue of human Htt
Oxygen dissociation from ferrous oxygenated human hemoglobin:haptoglobin complexes confirms that in the R-state α and β chains are functionally heterogeneous.
Full text linkThe adverse effects of extra-erythrocytic hemoglobin (Hb) are counterbalanced by several plasma proteins devoted to facilitate the clearance of free heme and Hb. In particular, haptoglobin (Hp) traps the αβ dimers of Hb, which are delivered to the reticulo-endothelial system by CD163 receptor-mediated endocytosis. Since Hp:Hb complexes show heme-based reactivity, kinetics of O2 dissociation from the ferrous oxygenated human Hp1-1:Hb and Hp2-2:Hb complexes (Hp1-1:Hb(II)-O2 and Hp2-2:Hb(II)-O2, respectively) have been determined. O2 dissociation from Hp1-1:Hb(II)-O2 and Hp2-2:Hb(III)-O2 follows a biphasic process. The relative amplitude of the fast and slow phases ranges between 0.47 and 0.53 of the total amplitude, with values of koff1 (ranging between 25.6 ± 1.4 s-1 and 29.1 ± 1.3 s-1) being about twice faster than those of koff2 (ranging between 13.8 ± 1.6 s-1 and 16.1 ± 1.2 s-1). Values of koff1 and koff2 are essentially the same independently on whether O2 dissociation has been followed after addition of a dithionite solution or after O2 displacement by a CO solution in the presence of dithionite. They correspond to those reported for the dissociation of the first O2 molecule from tetrameric Hb(II)-O2, indicating that in the R-state α and β chains are functionally heterogeneous and the tetramer and the dimer behave identically. Accordingly, the structural conformation of the α and β chains of the Hb dimer bound to Hp corresponds to that of the subunits of the Hb tetramer in the R-state
Modelling of the citrus ccd4 family members: In silico analysis of membrane binding and substrate preference
Full text linkColoring is one of the most important characteristics in commercial flowers and fruits, generally due to the accumulation of carotenoid pigments. Enzymes of the CCD4 family in citrus intervene in the generation of β-citraurin, an apocarotenoid responsible for the reddish-orange color of mandarins. Citrus CCD4s enzymes could be capable of interacting with the thylakoid membrane inside chloroplasts. However, to date, this interaction has not been studied in detail. In this work, we present three new complete models of the CCD4 family members (CCD4a, CCD4b, and CCD4c), modeled with a lipid membrane. To identify the preference for substrates, typical carotenoids were inserted in the active site of the receptors and the protein–ligand interaction energy was evaluated. The results show a clear preference of CCD4s for xanthophylls over aliphatic carotenes. Our findings indicate the ability to penetrate the membrane and maintain a stable interaction through the N-terminal α-helical domain, spanning a contact surface of 2250 to 3250 Å2. The orientation and depth of penetration at the membrane surface suggest that CCD4s have the ability to extract carotenoids directly from the membrane through a tunnel consisting mainly of hydrophobic residues that extends up to the catalytic center of the enzyme
Role of the electrostatic loop charged residues in Cu,Zn superoxide dismutase
No full textWe have expressed and characterized a mutant of Xenopus laevis Cu,Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lps120 --> Leu, Asp130 --> Gin, Glu131 --> Gin, and Lys134 --> Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6.7 x 10(9) M-1 s(-1), at pH 7 and mu = 0.02 M). Brownian dynamics simulations give rise to identical enzyme-substrate association rates for both wild-type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild-type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site
Cytochrome c: An extreme multifunctional protein with a key role in cell fate
No full textCytochrome c, a protein that belongs to class 1 of the c-type cytochrome family, exerts different functions depending on its cellular localization and the conditions in which it operates; therefore, it can be defined as ‘extreme multifunctional’ protein. It mediates electron-transfer in the respiratory chain and acts as a detoxifying agent to dispose of ROS. In addition, cytochrome c plays a role in cell apoptosis. After its release into the cytosol, the protein binds to APAF-1, activates pro-caspase 9, and triggers an enzymatic cascade leading to cell death. The interaction with cardiolipin, one of the phospholipids making up the mitochondrial membrane, is essential to start apoptosis; the binding partially unfolds cytochrome c, alters the heme pocket region, and facilitates detachment of Met80 from the sixth coordination position of the heme iron. These events change the function of cytochrome c from an electron-transfer shuttle to a peroxidase-like hemoprotein, capable to trigger the process that leads to cell death. This review provides an overview of the key role played by the cytochrome c-cardiolipin interaction in apoptosis. This is not only important per se, it provides interesting perspectives for applications in clinical diagnostics that use the protein as a biomarker
GA/GB fold switching may modulate fatty acid transfer from human serum albumin to bacteria.
No full textCeruloplasmin-ferroportin system of iron traffic in vertebrates
No full textSafe trafficking of iron across the cell membrane is a delicate process that requires specific protein carriers. While many proteins involved in iron uptake by cells are known, only one cellular iron export protein has been identified in mammals: ferroportin (SLC40A1). Ceruloplasmin is a multicopper enzyme endowed with ferroxidase activity that is found as a soluble isoform in plasma or as a membrane-associated isoform in specific cell types. According to the currently accepted view, ferrous iron transported out of the cell by ferroportin would be safely oxidized by ceruloplasmin to facilitate loading on transferrin. Therefore, the ceruloplasmin-ferroportin system represents the main pathway for cellular iron egress and it is responsible for physiological regulation of cellular iron levels. The most recent findings regarding the structural and functional features of ceruloplasmin and ferroportin and their relationship will be described in this review
Identification of the residues responsible for the alkaline inhibition of Cu,Zn superoxide dismutase: A site-directed mutagenesis approach
No full textThe catalytic rate of wild type, two single (Lys 120 --> Leu, Lys 134 --> Thr), and one double (Lys 120 --> Leu-Lys 134 --> Thr) mutants of Xenopus laevis B Cu,Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the activity of the wild-type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK(1)) and at pH 11.3 (pK(2)) Catalytic rate measurements on single and double mutants indicate that pK, is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK(2) is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper-bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141
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