Recessive mutations in DOCK6, encoding the guanidine nucleotide exchange factor DOCK6, lead to abnormal actin cytoskeleton organization and Adams-Oliver syndrome

Adams-Oliver syndrome (AOS) is defined by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It is usually inherited as an autosomal-dominant trait, but autosomal-recessive inheritance has also been documented. In an individual with autosomal-recessive AOS, we combined autozygome analysis with exome sequencing to identify a homozygous truncating mutation in dedicator of cytokinesis 6 gene (DOCK6) which encodes an atypical guanidine exchange factor (GEF) known to activate two members of the Rho GTPase family: Cdc42 and Rac1. Another homozygous truncating mutation was identified upon targeted sequencing of DOCK6 in an unrelated individual with AOS. Consistent with the established role of Cdc42 and Rac1 in the organization of the actin cytoskeleton, we demonstrate a cellular phenotype typical of a defective actin cytoskeleton in patient cells. These findings, combined with a Dock6 expression profile that is consistent with an AOS phenotype as well as the very recent demonstration of dominant mutations of ARHGAP31 in AOS, establish Cdc42 and Rac1 as key molecules in the pathogenesis of AOS and suggest that other regulators of these Rho GTPase proteins might be good candidates in the quest to define the genetic spectrum of this genetically heterogeneous condition.     

DOCK 6: Combining techniques to model RNA–small molecule complexes

With an increasing interest in RNA therapeutics and for targeting RNA to treat disease, there is a need for the tools used in protein-based drug design, particularly DOCKing algorithms, to be extended or adapted for nucleic acids. Here, we have compiled a test set of RNA–ligand complexes to validate the ability of the DOCK suite of programs to successfully recreate experimentally determined binding poses. With the optimized parameters and a minimal scoring function, 70% of the test set with less than seven rotatable ligand bonds and 26% of the test set with less than 13 rotatable bonds can be successfully recreated within 2 Å heavy-atom RMSD. When DOCKed conformations are rescored with the implicit solvent models AMBER generalized Born with solvent-accessible surface area (GB/SA) and Poisson–Boltzmann with solvent-accessible surface area (PB/SA) in combination with explicit water molecules and sodium counterions, the success rate increases to 80% with PB/SA for less than seven rotatable bonds and 58% with AMBER GB/SA and 47% with PB/SA for less than 13 rotatable bonds. These results indicate that DOCK can indeed be useful for structure-based drug design aimed at RNA. Our studies also suggest that RNA-directed ligands often differ from typical protein–ligand complexes in their electrostatic properties, but these differences can be accommodated through the choice of potential function. In addition, in the course of the study, we explore a variety of newly added DOCK functions, demonstrating the ease with which new functions can be added to address new scientific questions.     

Identification of DOCK4 and its splicing variant as PIP3 binding proteins

DOCK4, a member of DOCK180 family proteins, was originally identified as a product of a gene deleted during tumor progression. Although its tumor suppression properties have been reported, the regulation mechanism of this protein has not been fully elucidated. DOCK4 shares two conserved domains called as DHR-1 and DHR-2 domain as other members including DOCK180. Although DHR-1 in DOCK180 is reported to bind to PIP(3), whether that of DOCK4 exhibits similar function has yet not been examined. In a search for novel PIP(3) binding proteins by the PIP(3) analog beads binding assay, we found that DOCK4 and its novel splicing variant, whose exon1 and exon52 are different from the known one, bind to PIP(3). Binding assay using deletion mutants of DOCK4 revealed that the binding region falls into the DHR-1 domain. These results raise the possibility that DOCK4 may be regulated by PIP(3) to exert its function.     

Altered frequency-dependent inactivation and steady-state inactivation of polyglutamine-expanded alpha1A in SCA6

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease of the cerebellum and inferior olives characterized by a late-onset cerebellar ataxia and selective loss of Purkinje neurons (15, 16). SCA6 arises from an expansion of the polyglutamine tract located in exon 47 of the _1A (P/Q-type calcium channel) gene from a nonpathogenic size of 4 to 18 glutamines (CAG_4–18) to CAG_19–33 in SCA6. The molecular basis of SCA6 is poorly understood. To date, the biophysical properties studied in heterologous systems support both a gain and a loss of channel function in SCA6. We studied the behavior of the human _1A isoform, previously found to elicit a gain of function in disease (41), focusing on properties in which the COOH terminus of the channel is critical for function: we analyzed the current properties in the presence of ₄- and _2a-subunits (both known to interact with the _1A COOH terminus), current kinetics of activation and inactivation, calcium-dependent inactivation and facilitation, voltage-dependent inactivation, frequency dependence, and steady-state activation and inactivation properties. We found that SCA6 channels have decreased activity-dependent inactivation and a depolarizing shift (+6 mV) in steady-state inactivation properties consistent with a gain of function. trinucleotide repeats; ataxia; calmodulin     

Metabolomic profiling highlights oxidative damages in platelet concentrates treated for pathogen inactivation and shows protective role of urate

Introduction

Research in transfusion medicine is motivated by the desire to deliver the most-compatible and most-efficient blood product to the patient. It is now well accepted that ex vivo platelet concentrates (PCs) experience biochemical alterations and a functional decline known as storage lesions. Photochemical treatments have been introduced to secure PCs against pathogens but are reported to accelerate these lesions.

Objectives

The objective of this study was to investigate metabolic changes in stored PCs treated for pathogen inactivation with the INTERCEPT Blood system (Cerus, Concord, USA).

Methods

PCs either untreated (uPCs) or INTERCEPT-treated (iPCs) were sampled along the 7-day storage period. First, metabolites were extracted and analyzed using ultra-high pressure liquid chromatography—high resolution mass spectrometry followed by statistical analysis. Secondly, we investigated the role of urate, a major plasma antioxidant, in the platelet function using flow cytometry-based assays.

Results

We observed oxidative damages in stored iPCs compared to uPCs, in particular alteration of the purine and the glutathione metabolism. We showed diminution of antioxidant defenses following INTERCEPT treatment such as the conversion of urate to allantoin, only possible in humans under the action of reactive oxygen species (ROS). Functional assays on platelets in absence or in an excess of urate suggest a protective role of urate in PCs.

Conclusion

Our results indicate oxidative damages occurring at the metabolic level in stored iPCs. Understanding better the role of antioxidants such as urate in ex vivo PCs would definitively provide new insights to ameliorate the storage conditions and preserve the functionality of platelets.

     

Hippocampal gene expression analysis highlights Ly6a/Sca-1 as candidate gene for previously mapped novelty induced behaviors in mice

In this study, we show that the covariance between behavior and gene expression in the brain can help further unravel the determinants of neurobehavioral traits. Previously, a QTL for novelty induced motor activity levels was identified on murine chromosome 15 using consomic strains. With the goal of narrowing down the linked region and possibly identifying the gene underlying the quantitative trait, gene expression data from this F(2)-population was collected and used for expression QTL analysis. While genetic variation in these mice was limited to chromosome 15, eQTL analysis of gene expression showed strong cis-effects as well as trans-effects elsewhere in the genome. Using weighted gene co-expression network analysis, we were able to identify modules of co-expressed genes related to novelty induced motor activity levels. In eQTL analyses, the expression of Ly6a (a.k.a. Sca-1) was found to be cis-regulated by chromosome 15. Ly6a also surfaced in a group of genes resulting from the network analysis that was correlated with behavior. Behavioral analysis of Ly6a knock-out mice revealed reduced novelty induced motor activity levels when compared to wild type controls, confirming functional importance of Ly6a in this behavior, possibly through regulating other genes in a pathway. This study shows that gene expression profiling can be used to narrow down a previously identified behavioral QTL in mice, providing support for Ly6a as a candidate gene for functional involvement in novelty responsiveness.     

The DOCK180/Elmo complex couples ARNO-mediated Arf6 activation to the downstream activation of Rac1

Cell motility requires extensions of the plasma membrane driven by reorganization of the actin cytoskeleton. Small GTPases, particularly the Rho family, are key regulators of this process. A second class of GTPases, the ADP-ribosylation factors (ARFs), have also been implicated in the regulation of the actin cytoskeleton and motility. ARF6 is intimately involved in the regulation of Rac activity; however, the mechanisms by which ARF activation leads to activation of Rac remain poorly understood. We have previously shown that expression of the ARF-GEF ARNO in MDCK cells induces robust activation of Rac, the formation of large lamellipodia, and the onset of motility. We report here that ARNO-dependent activation of Rac is mediated by a bipartite Rac GEF, the Dock180/Elmo complex. Both DOCK180 and Elmo colocalize extensively with ARNO in migrating MDCK cells. Importantly, both a catalytically inactive Dock180 mutant and an Elmo mutant that fails to couple to Dock180 block ARNO-induced Rac activation and motility. In contrast, a similar mutant of the Rac GEF beta-PIX fails to inhibit ARNO-induced Rac activation or motility. Together, these data suggest that ARNO and ARF6 coordinate with the Dock180/Elmo complex to promote Rac activation at the leading edge of migrating cells.     

alpha-crystallin protects glucose 6-phosphate dehydrogenase against inactivation by malondialdehyde

The present work investigates the effect of malondialdehyde (MDA) binding on the enzymic activity and on some structural properties of glucose 6-phosphate dehydrogenase (G6PD). We studied whether alpha-crystallin could protect the enzyme against MDA damage, and if so, by what mechanism. We also studied whether alpha-crystallin could renature G6PD denatured by MDA. alpha-Crystallin was prepared from bovine lenses by gel chromatography. MDA was freshly prepared and incubated with G6PD with or without alpha-crystallin. The results show that MDA reacted with G6PD non-enzymically causing inactivation at concentrations lower than those used previously on structural proteins. The modified enzyme became fluorescent. alpha-Crystallin, acting as a molecular chaperone, specifically protected the enzyme against inactivation by MDA. The enzyme was not reactivated by alpha-crystallin, but it was stabilised and protected against further denaturation. Complex formation between alpha-crystallin and the modified enzyme was demonstrated by immunoprecipitation. G6PD was very susceptible to MDA and we have shown for the first time that alpha-crystallin is able to protect the enzyme against this damage.     

Drought-induced activation and rehydration-induced inactivation of MPK6 in Arabidopsis

Mitogen-activated protein kinases (MPKs) have roles in regulating developmental processes and responses to various stimuli in plants. Activations of some MPKs are necessary for proper responses to hyperosmolarity and to a stress-related phytohormone, abscisic acid (ABA). However, there is no direct evidence that MPK activations are regulated by drought and rehydration. Here we show that the activation state of one of the Arabidopsis MPKs, MPK6, is directly regulated by drought and rehydration. An immunoblot analysis using an anti-active MPK antibody detected drought-induced activation and rehydration-induced inactivation of MPK6. MPK6 was activated by drought even in an ABA-deficient mutant, aba2-4. In addition, exogenously added ABA failed to suppress the rehydration-dependent inactivation of MPK6. Under drought conditions, elevated levels of reactive oxygen species (ROS), which are known elicitors of MPK6 activation, were detected in both wild type and an MPK6-deficient mutant, mpk6-4. These results suggest that ROS, but not ABA, induces MPK6 activation as an upstream signal under drought conditions.     

Mechanism-based inactivation of rabbit muscle phosphoglucomutase by nojirimycin 6-phosphate

Nojirimycin 6-phosphate (N6P) was tested as a substrate and inhibitor for phosphoglucomutase (PGM). In the absence of glucose 1,6-bisphosphate (GBP), the incubation of PGM and N6P resulted in the complete inactivation of all enzyme activity. When equimolar amounts of N6P and GBP were incubated together with PGM, the GBP was quantitatively converted to glucose 6-phosphate (G6P) and phosphate. At higher ratios of GBP and N6P (greater than 100) the final concentration of G6P produced was found to be 19 times the initial N6P concentration. These results have been interpreted to suggest that the phosphorylated form of PGM catalyzes the phosphorylation of N6P at C-1. This intermediate rapidly eliminates phosphate to form an imine and the dephosphorylated enzyme. The dephosphorylated enzyme is rapidly rephosphorylated by GBP and forms G6P. The imine is nonenzymatically hydrated back to N6P. Occasionally (5%) the imine isomerizes to a compound that is not processed by PGM.     

Sodium channel opening as a precursor to inactivation

The time constant of the process producing the delay in Na inactivation development as determined by the two pulse method (_delay) was extracted and compared to that of the slowest Na activation process ₃ for the I _Na during the conditioning pulse of that same determination. _delay and two pulse inactivation _c values were computer generated using a nonlinear least squares algorithm. _h and single pulse inactivation _h values were independently generated for each determination also with the aid of the computer using the same non-linear least squares algorithm. In one determination at 2 mV, _c was 4.68 and _delay 0.494 ms while _h was 4.70 and ₃ 0.491 ms for a _c/_h of 0.996 and a _delay/₃ of 1.006. Mean _delay/₃ from five determinations in four axons, both Cs and K perfused, and spanning a potential range of-27 to 2mV was 1.068. The precursor process to inactivation is channel opening. Some fraction of channels presumably inactivate via another route where prior channel opening is not required.     

Radiation inactivation analysis of rat liver microsomal glucose-6-phosphatase

Radiation inactivation analysis was utilized to estimate the sizes of the units catalyzing the various activities of hepatic microsomal glucose-6-phosphatase. This technique revealed that the target molecular weights for mannose-6-P phosphohydrolase, glucose-6-P phosphohydrolase, and carbamyl-P:glucose phosphotransferase activities were all about Mr 75,000. These results are consistent with the widely held view that all of these activities are catalyzed by the same protein or proteins. Certain observations indicate that the molecular organization of microsomal glucose-6-phosphatase is better described by the conformational hypothesis which envisions the enzyme as a single covalent structure rather than by the substrate transport model which requires the participation of several physically separate polypeptides. These include the findings: 1) that the target sizes for glucose-6-P phosphohydrolase and carbamyl-P:glucose phosphotransferase activities were not larger than that for mannose-6-P phosphohydrolase in intact microsomes and 2) that the target size for glucose-6-P phosphohydrolase in disrupted microsomes was not less than that observed in intact microsomes. These findings are most consistent with a model for glucose-6-phosphatase of a single polypeptide or a disulfide-linked dimer which spans the endoplasmic reticulum with the various activities of this multifunctional enzyme residing in distinct protein domains.     

Inhibition by aldehydes as a possible further mechanism for glucose-6-phosphatase inactivation during CCl4-poisoning

Diffusable aldehydes are known to be produced during lipoperoxidative deterioration of unsaturated fatty acids. Malealdehyde (MLA) and 4-hydroxy-2,3-trans-penten-1-al (4-HPE) inhibit rat liver glucose-6-phosphatase activity in vitro. With MLA inhibition is significant at 0.25 mM concentration. With 4-HPE inhibition takes place at 0.5 mM. 1 mM MLA inhibited by about 89%, 6 mM 4-HPE by about 67%. Maximal inhibition is present as early as 5 min after addition of both aldehydes. Preincubation of aldehydes with 2 mM cysteine or glycine in the absence of microsomes almost completely prevents the inhibitory influence. Previous incubation of microsomes with 2 mM glutathione or 2 mM dithiothreitol or 2 mM cysteine affords a good protection towards the inhibitory action of the aldehydes; on the contrary, no protection is seen when microsomes are preincubated in the presence of either 2 mM glycine or asparagine. The total content of microsomes -SH groups is strongly decreased after incubation with 2mM malealdehyde.These results support the idea that the two aldehydes inhibit glucose-6-phosphatase mostly through interaction with protein -SH groups. The possibility that aldehydes derivated from the peroxidative decomposition of lipids may play a cooperative role in the inhibition of glucose-6-phosphatase occurring early after CCl₄-poisoning is discussed.     

The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.     

Kinetics of inactivation of beta-lactamase I by 6 beta-bromopenicillanic acid.

The kinetics of the inactivation of beta-lactamase I from Bacillus cereus 569 by preparations of 6 alpha-bromopenicillanic acid showed unexpected features. These can be quantitatively accounted for on the basis of the inactivator being the epimer, 6 beta-bromopenicillanic acid. At pH 9.2, the rate-determining step in the inactivation is the formation of the inactivator. When pure 6 beta-bromopenicillanic acid is used to inactivate beta-lactamase I, simple second-order kinetics are observed. The inactivated enzyme has a new absorption peak at 326 nm. The rate constant for inactivation has the same value as the rate constant for appearance of absorption at 326 nm; the rate-determining step may thus be fission of the beta-lactam ring of 6 beta-bromopenicillanic acid. Inactivation is slower in the presence of substrate, and the observed kinetics can be quantitatively accounted for on a simple competitive model. The results strongly suggest that inactivation is a consequence of reaction at the active site.     

Suicide inactivation of the E. coli O6-methylguanine-DNA methyltransferase

The O6-methylguanine-DNA methyltransferase of Escherichia coli acts rapidly and stoichiometrically to convert a mutagenic O6-methylguanine residue in DNA to unsubstituted guanine. Even at low protein concentrations and in the absence of any cofactors, the transfer of a methyl group to one of the protein's own cysteine residues occurs in less than 2 s at 37 degrees C. The entire kinetic process can be followed experimentally at 5 degrees C. Formation of S-methylcysteine in the protein is accompanied by loss of activity and accounts for the exceptional suicide kinetics of this enzyme as well as for the sharp saturation of O6-methylguanine repair observed in vivo. The enzyme can remove greater than 98% of the methyl groups from O6-methylguanine present in alkylated DNA, but leaves N-alkylated purines untouched. Single-stranded DNA containing O6-methylguanine is a poor substrate, with the methyl transfer occurring at approximately 0.1% of the rate for duplex DNA. This latter observation may explain the high frequency of mutations induced by alkylating agents at DNA replication forks.     

Mechanisms of inactivation of human S-adenosylhomocysteine hydrolase by 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosines

In an effort to design more specific and potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, we investigated the mechanisms by which 5',5',6', 6'-tetradehydro-6'-deoxy-6'-halohomoadenosines (X = Cl, Br, I) inactivated this enzyme. The 6'-chloro (a) and 6'-bromo (b) acetylenic nucleoside analogues produced partial ( approximately 50%) loss of enzyme activity with a concomitant ( approximately 50%) reduction of E-NAD(+) to E-NADH. In addition, Ade and halide ions were released from the inhibitors in amounts suggestive of a process involving enzyme catalysis. AdoHcy hydrolase, which was inactivated with compound a, was shown to contain 2 mol of the inhibitor covalently bound to Lys318 of two subunits of the homotetramer. These data suggest that the enzyme-mediated water addition at the 5' position of compound a or b produces an alpha-halomethyl ketone intermediate, which is then attacked by a proximal nucleophile (i.e., Lys318) to form the enzyme-inhibitor covalent adduct (lethal event); in a parallel pathway (nonlethal event), addition of water at the 6' position produces an acyl halide, which is released into solution and chemically degrades into Ade, halide ion, and sugar-derived products. In contrast, compound c completely inactivated AdoHcy hydrolase by converting 2 equiv of E-NAD(+) to E-NADH and causing the release of 2 equiv of E-NAD(+) into solution. Four moles of the inhibitor was shown to be tightly bound to the tetrameric enzyme. These data suggest that compound c inactivates AdoHcy hydrolase by a mechanism similar to the acetylenic analogue of Ado described previously by Parry et al. [(1991) Biochemistry 30, 9988-9997].     

alphaA- and alphaB-crystallins protect glucose-6-phosphate dehydrogenase against UVB irradiation-induced inactivation

alpha-Crystallin, a major eye lens protein, has been shown to function like a molecular chaperone by suppressing the aggregation of other proteins induced by various stress conditions. Ultraviolet (UV) radiation is known to cause structural and functional alterations in the lens macromolecules. Earlier we observed that exposure of rat lens to in vitro UV radiation led to inactivation of many lens enzymes including glucose-6-phosphate dehydrogenase (G6PD). In the present paper, we show that alpha-crystallin (alphaA and alphaB) protects G6PD from UVB irradiation induced inactivation. While, at 25 degrees C, there was a time-dependent decrease in G6PD activity upon irradiation at 300 nm, at 40 degrees C there was a complete loss of activity within 30 min even without irradiation. The loss of activity of G6PD was prevented significantly, if alphaA- or alphaB-crystallin was present during irradiation. At 25 degrees C, alphaB-crystallin was slightly a better chaperone in protecting G6PD against UVB inactivation. Interestingly, at 40 degrees C, alphaA- and alphaB-crystallins not only prevent the loss of G6PD activity but also protect against UVB inactivation. However, alphaA- and alphaB-crystallins were equally efficient at 40 degrees C in protecting G6PD.