Modulation of mitochondrial succinate dehydrogenase activity,mechanism and function

Summary The mitochondrial succinate dehydrogenase (E.C. 1.3.3.99) is subjected to apparently complicated regulatory mechanism. Yet, systematic analysis of the mechanism reveals the simplicity of the control. There are two stable forms of the enzyme; the non-active form stabilized as 1:1 complex with oxaloacetate and the active form stabilized by binding of activating ligands. This model quantitatively describes either the equilibrium level of active enzyme or the kinetics of activation-deactivation, in the presence of various concentrations of opposing effectors. The site where the regulatory ligands interact with the enzyme is not the substrate bonding site. The marked differences of dissociation constants of the same ligand from the two sites clearly distinguish between them.This model is fully developed for simple cases where the activating ligands are dicarboxylic acids or monovalent anions. On the other hand with activators such as ATP or CoQH₂, quantitation is still not at hand. This stems from the difficulties in maintaining determined, measurable, concentrations of the ligand in equilibrium with the membranal enzyme.While in active form the histidyl flavin moity of the enzyme is reduced by physiological substrate (succinate; CoQH₂). The non-active form is not reduced by these compounds, only strong reductants with low redox potential reduce the non-active enzyme. It is suggested that deactivation is a simple modulation of the redox potential of the flavin form E 0 mV in the active enzyme to E < –190 mV. The switch from one state to another might be achieved by distortion of the planar form of oxidized flavin to the bend configuration of the reduced flavin. Thus, in the active enzyme such distortion will destabilize the oxidized state of the flavin, shifting the redox potential to the higher value. The binding of oxaloacetate to the regulatory sites releases the distorting forces by relaxing the conformation of the enzyme. Consequently, the flavin assumes its planar form with the low redox potential. This assumption is supported by the spectral shifts of the flavin associated with the activation deactivation transition.The suicidal oxidation of malate to oxaloacetate, carried by the succinate dehydrogenase, plays an important role in modulating the enzyme activity in the mitochondria. This mechanism might supply oxaloacetate for deactivation in spite of the negligible concentration of free oxaloacetate in the matrix. The oxidation of malate by the enzyme is controlled by the redox potential at the immediate vicinity of the enzyme, and is imposed by the redox level of the membranal quinone.Finally, the modulation of succinate dehydrogenase activity is closely associated with regulation of NADH oxidation through the mutual inhibition between oxidases (Gutman, M. in Bioenergetics of Membranes, L. Packer et al., ed. Elsevier 1977, p. 165). The consequence of these interactions is the selection for the main electron donnor for the respiratory chain, during mixed substrate respiration, according to the metabolic demands from the mitochondria.Abbreviations SDH succinate dehydrogenase (succinate: acceptor oxidoreductase (E.C. 1.3.99.1)); - OAA oxaloacetate - Act activator - E_A, E_A active and non active forms of the enzyme, respectively - K'eq apparent equilibrium constant - K'd apparent dissociation constant - K_Act, K_OAA dissociation constant of the respective ligand from the enzyme - K'a, k'd the apparent rate constants of activation and deactivation, respectively - ka, kd the true rate constant of activation and deactivation respectively - ETP, ETP_II non phosphorylating and phosphorylating submitochondrial particles - PMS phenazine methosulfate - DCIP dichlorophenol indophenol - CoQ ubiquinone - TIFA Thenotriflouvoacetone - NEM N methyl Maleimide     

Prenatal diagnosis of familial hypercholesterolemia caused by the “Lebanese” mutation at the low density lipoprotein receptor locus

Summary Here, we report the prenatal diagnosis of familial hypercholesterolemia in a Christian-Arab family that carries the Lebanese mutation, a single base substitution that creates a HinfI restriction site, at the low density lipoprotein (LDL) receptor locus. Polymerase chain reaction amplification and restriction analysis were performed on genomic DNA extracted from a chorionic villus sample. In conjunction with karyotype analysis, the fetus was identified as a heterozygous female. Analysis of LDL receptor restriction fragment length polymorphisms confirmed the presence of a male parent marker and revealed that the fetus inherited the mutant gene from its mother. This technique offers a simple and rapid diagnostic tool that can be carried out at an early stage of gestation. It is recommended for families and population groups with molecularly defined LDL receptor mutations.     

Cerebrospinal Fluid Parameters in Healthy Volunteers During Serial Lumbar Punctures

Lumbar punctures were performed on four occasions over a 5-day period (8:30 a.m. on days 1, 3, and 5; 2:30 p.m. on day 2) on 10 normal volunteers (five of each sex; mean age, 27.7 years) to assess, with repeated sampling, the day-to-day variation of selected CSF parameters. Two subjects abstained from the lumbar puncture on day 5 due to headache after the third puncture. Lumbar CSF was analyzed for concentrations of free and total gamma-aminobutyric acid (GABA), homocarnosine, homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), total protein, albumin, and immunoglobulin (Ig)G. No significant concentration differences were found between the afternoon and next morning samples. No differences were found in concentrations of free GABA, total GABA, homocarnosine, 5-HIAA, or albumin across the study. In contrast, HVA concentrations significantly increased by day 5, whereas total protein and IgG decreased during the study. The most likely explanation for these changes involves the known concentration gradients in the CSF column.     

A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling

Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c-Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c-Cbl, here we report that defective Tyr1045 of EGFR, an inducible c-Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand-induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization-defective mutant receptor imply that c-Cbl-mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand-induced ubiquitylation, especially in the presence of an overexpressed c-Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c-Cbl complex to Grb2-specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well-characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c-Cbl-dependent sorting of active tyrosine kinases to destruction.     

Cytochalasin D disruption of actin filaments in 3T3 cells produces an anti-apoptotic response by activating gelatinase A extracellularly and initiating intracellular survival signals

Disruption of actin filaments affects multiple cell functions including motility, signal transduction and cell division, ultimately culminating in cell death. Although this is the usual sequence of events, we have made the interesting observation that disruption of actin filaments by the potent toxin cytochalasin D (Cyto D) causes one cell type, mouse mesangial cells (MMC), to undergo apoptosis, while in another cell type (NIH 3T3), it has the opposite effect, resulting in production of survival signals. The purpose of this study was to investigate the molecular basis for these observed differences. In the present communication, we demonstrate that exposure to Cyto D induces the pro-apoptotic pathways, p38 and stress-activated protein kinase (SAPK)/jun amino-terminal kinase (JNK), in both cell types. However, in 3T3, but not MMC, the extracellular signal regulated kinase (ERK) 1/2 pathway is protected from inhibition following treatment with Cyto D-leading to phosphorylation of Bclxi/Bcl 2-associated death promoter (BAD). Inhibition of Cyto D-induced secretion and activation of gelatinase A in 3T3 cells reverses the production of survival signals by Cyto-D. To investigate this effect further we employed CS-1 cells, a well-characterized melanoma cell line that lacks integrin beta3, and also does not secrete gelatinase A. Co-transfection of CS-1 cells with integrin beta3 and a gelatinase A transgene, which enables the cells to secrete constituitively active gelatinase A, enhances CS-1 cell survival signals. Together, our findings suggest that extracellularly activated gelatinase A, through interaction with integrin alphaVbeta3, elicits survival signals mediated through ERK 1/2 that override activation of p38 and SAPK/JNK stress pathways.     

Ligand-independent degradation of epidermal growth factor receptor involves receptor ubiquitylation and Hgs,an adaptor whose ubiquitin-interacting motif targets ubiquitylation by Nedd4

Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.     

Dynamic changes in the osmotic water permeability of protoplast plasma membrane

The osmotic water permeability coefficient (P(f)) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The best-fitting model had three free parameters: initial P(f), P(f) rate-of-change (slope(P(f))), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The P(f) during the delay was < or =1 microm s(-1). During the swelling period following the delay, P(f) changed dynamically: within the first 15 s P(f) either (1) increased gradually to approximately 8 microm s(-1) (in the majority population of low-initial-P(f) cells) or (2) increased abruptly to 10 to 20 microm s(-1) and then decreased gradually to 3 to 6 microm s(-1) (in the minority population of high-initial-P(f) cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial P(f) values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the P(f) in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing P(f) increase, thereby confirming the hypothesis that P(f) dynamics, delay included, reflected the varying activity of aquaporins.     

A functional hierarchical organization of the protein sequence space

Background

It is a major challenge of computational biology to provide a comprehensive functional classification of all known proteins. Most existing methods seek recurrent patterns in known proteins based on manually-validated alignments of known protein families. Such methods can achieve high sensitivity, but are limited by the necessary manual labor. This makes our current view of the protein world incomplete and biased. This paper concerns ProtoNet, a automatic unsupervised global clustering system that generates a hierarchical tree of over 1,000,000 proteins, based solely on sequence similarity.

Results

In this paper we show that ProtoNet correctly captures functional and structural aspects of the protein world. Furthermore, a novel feature is an automatic procedure that reduces the tree to 12% its original size. This procedure utilizes only parameters intrinsic to the clustering process. Despite the substantial reduction in size, the system's predictive power concerning biological functions is hardly affected. We then carry out an automatic comparison with existing functional protein annotations. Consequently, 78% of the clusters in the compressed tree (5,300 clusters) get assigned a biological function with a high confidence. The clustering and compression processes are unsupervised, and robust.

Conclusions

We present an automatically generated unbiased method that provides a hierarchical classification of all currently known proteins.