Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate,cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism

The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis. Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy. The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves. Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme. It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists. The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+). In addition, a potassium ion has been located at the dimer interface. A structural model for the kinetic mechanism is proposed.     

A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells

Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for reasons that are unknown. Here, we sought to provide some foundational information on Impdh1a in the zebrafish retina. We found that in zebrafish, gene subfunctionalization due to ancestral duplication resulted in a predominant retinal variant expressed exclusively in rod and cone photoreceptors. This variant is structurally and functionally similar to the human IMPDH1 retinal variant and shares a reduced sensitivity to GTP-mediated inhibition. We also demonstrated that Impdh1a forms prominent protein filaments in vitro and in vivo in both rod and cone photoreceptor cell bodies, synapses, and to a lesser degree, in outer segments. These filaments changed length and cellular distribution throughout the day consistent with diurnal changes in both mRNA and protein levels. The loss of Impdh1a resulted in a substantial reduction of guanine levels, although cellular morphology and cGMP levels remained normal. Our findings demonstrate a significant role for IMPDH1 in photoreceptor guanine production and provide fundamental new information on the details of this protein in the zebrafish retina.     

Identification and characterization of the unique guanine nucleotide exchange factor, SmgGDS, in vascular smooth muscle cells

The guanine nucleotide exchange factor (GEF), SmgGDS, promotes nucleotide exchange by several GTPases in both the Ras and Rho families, especially by RhoA. Because RhoA plays an important role in regulating the contraction of vascular smooth muscle cells (VSMC), we examined the expression and function of SmgGDS in VSMC. SmgGDS is expressed in primary rat aortic smooth muscle (ASM) cells, primary bovine coronary artery smooth muscle (BCASM) cells, and the immortalized A7r5 line of rat ASM cells. Down regulation of SmgGDS expression by siRNA transfection resulted in a decrease of RhoA-GTP levels, enhanced cell spreading, and loss of the characteristic elongated morphology of VSMC. A similar morphology was also observed following treatment with the Rho-kinase inhibitor, Y27632. In contrast, cells with reduced RhoA expression exhibit an elongated shape. Subsequent immunofluorescent staining revealed a disruption of the myosin filament organization in the cells with reduced SmgGDS expression. Further studies analyzed the effect of SmgGDS siRNA transfection on the contraction of A7r5 cells and BCASM cells, which is also a Rho-regulated pathway. Transfection of SmgGDS siRNA or RhoA siRNA resulted in an impaired ability of the A7r5 and BCASM cells to undergo contraction in a collagen gel matrix. However, phosphorylation of the myosin-binding subunit of myosin phosphatase (MYPT1) or the light chain of myosin II (MLC) was not altered by downregulating expression of either SmgGDS or RhoA GTPase. Taken together these results identify SmgGDS as a novel regulator of myosin organization and contraction in VSMC.     

CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.

T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.     

The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins

Guanine nucleotide exchange factors (GEFs) regulate the activity of small G proteins by catalysing the intrinsically slow exchange of GDP for GTP. The mechanism involves the formation of trimeric G protein-nucleotide-GEF complexes, followed by the release of nucleotide to form stable binary G protein-GEF complexes. A number of structural studies of G protein-GEF complexes have shown large structural changes induced in the nucleotide binding site. Together with a recent structure of a trimeric complex, these studies have suggested not only some common principles but also large differences in detail in the GEF-mediated exchange reaction. Several structures suggested that a glutamic acid residue in switch II, which is part of the DxxGQE motif and highly conserved in Ras-like G proteins, might have a decisive mechanistic role in GEF-mediated nucleotide exchange reactions. Here we show that mutation of the switch II glutamate to Ala severely impairs GEF-catalysed nucleotide exchange in most, but not all, Ras family G proteins, explaining its high sequence conservation. The residue determines the initial approach of GEF to the nucleotide-loaded G protein and does not appreciably affect the formation of a binary nucleotide-free complex. Its major effect thus appears to be the removal of the P-loop lysine from its interaction with the nucleotide.     

A concerted mechanism for opening the GDP binding pocket and release of the nucleotide in hetero-trimeric G-proteins

G-protein hetero-trimers play a fundamental role in cell function. Their dynamic behavior at the atomic level remains to be understood. We have studied the Gi hetero-trimer through a combination of molecular dynamics simulations and normal mode analyses. We showed that these big proteins could undergo large-amplitude conformational changes, without any energy penalty and with an intrinsic dynamics centered on their GDP binding pocket. Among the computed collective motions, one of the modes (mode 17) was particularly able to significantly open both the base and the phosphate sides of the GDP binding pocket. This mode describing mainly a motion between the Ras-like and the helical domains of G_α was in close agreement with some available X-ray data and with many other biochemical/biophysical observations including the kink of helix α5. The use of a new protocol, which allows extraction of the GDP ligand along the computed normal modes, supported that the exit of GDP was largely coupled to an opening motion along mode 17. We propose for the first time a “concerted mechanism” model in which the opening of the GDP pocket and the kink of the α5 helix occur concomitantly and favor GDP release from G_αβγ complexes. This model is discussed in the context of the G-protein-coupled receptor/G-protein interaction close to the cell membrane.     

An analysis of the role of guanine nucleotide binding proteins in antigen receptor/CD3 antigen coupling to phospholipase C.

In permeabilized human T lymphocytes, phospholipase C (PLC)-mediated metabolism of polyphosphatidylinositols can be stimulated by triggering the T cell antigen receptor/CD3 antigen complex (Ti/CD3) with the CD3 antibody UCHT1 or by activation of G proteins with the non-hydrolyzable guanine nucleotide analogue, guanosine 5'-O-(3-thiotrisphosphate) (GTP[S]). Ti/CD3 induction of inositol phosphate production demonstrated no dependence on exogenous guanine nucleotides. Furthermore, Ti/CD3 stimulation did not influence the kinetics or dose-response of GTP[S]-induced inositol phosphate production, suggesting that the Ti/CD3 complex does not regulate guanine nucleotide exchange on the G protein pool stimulated by GTP[S]. These data indicate that the Ti/CD3 complex is not G protein-linked to PLC in a manner analogous to the G protein linkage of receptors to adenylate cyclase. However, the inhibitory guanine nucleotide, GDP, antagonizes not only GTP[S]-induced polyphosphatidylinositol hydrolysis but also UCHT1-induced inositol phosphate production. These data infer that a G protein can modulate the coupling of the Ti/CD3 complex to PLC and that there may be some "cross-talk" between Ti/CD3 and G protein PLC coupling mechanisms.     

GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factors,is localized to the cis-Golgi and involved in membrane association of the COPI coat

Formation of coated carrier vesicles, such as COPI-coated vesicles from the cis -Golgi, is triggered by membrane binding of the GTP-bound form of ADP-ribosylation factors. This process is blocked by brefeldin A, which is an inhibitor of guanine nucleotide exchange factors for ADP-ribosylation factor. GBF1 is one of the guanine nucleotide-exchange factors for ADP-ribosylation factor and is localized in the Golgi region. In the present study, we have determined the detailed subcellular localization of GBF1. Immunofluorescence microscopy of cells treated with nocodazole or incubated at 15 °C has suggested that GBF1 behaves similarly to proteins recycling between the cis -Golgi and the endoplasmic reticulum. Immunoelectron microscopy has revealed that GBF1 localizes primarily to vesicular and tubular structures apposed to the cis -face of Golgi stacks and minor fractions to the Golgi stacks. GBF1 overexpressed in cells causes recruitment of class I and class II ADP-ribosylation factors onto Golgi membranes. Furthermore, overexpressed GBF1 antagonizes various effects of brefeldin A, such as inhibition of membrane recruitment of ADP-ribosylation factors and the COPI coat, and redistribution of Golgi-resident and itinerant proteins. These observations indicate that GBF1 is involved in the formation of COPI-coated vesicles from the cis -Golgi or the pre-Golgi intermediate compartment through activating ADP-ribosylation factors.     

Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian cells

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.     

The role of the nicotinamide and adenine subsites in the negative co-operativity of coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase.

The interaction of 3-aminopyridine-adenine dinucleotide, an NAD ⁺ 2 analogue which is fluorescent at the pyridine end of the molecule, with rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was investigated. The fluorescence properties of the AAD⁺ molecule were used to monitor the nicotinamide subsites ou the GPDHase tetramer, the fluorescent aminopyridine moiety of the molecule serving as an intrinsic probe. Although the binding of AAD⁺ wag found to be negatively co-operative, no conformational changes induced at the nicotinamide subsite upon coenzyme binding were found to be transmitted to neighboring subunits. These findings, in conjunction with our earlier findings and with the observation that different NAD⁺ analogues which differ in the chemistry of the pyridine moiety bind with different extents of co-operativity, enable us to offer specific roles for the nicotinamide and the adenine subsites in generating the negative co-operativity.It is suggested that the structure of the pyridine moiety of the coenzyme controls the mode of binding of the pyridine moiety to the nicotinamide subsite. This, in turn, controls the orientation of the adenine moiety with respect to its subsite, thereby determining the mode of the interactions between the adenine and its binding domain. As the propagation of conformational changes caused by these interactions to neighboring subunits is believed to be the cause of the negative co-operativity exhibited by this enzyme towards coenzyme binding, the structure of the pyridine moiety controls this phenomenon.     

A method for determining the in vivo stability of Drosophila alcohol dehydrogenase (E.C.1.1.1.1.)

A rapid and accurate method of measuring the relative in vivo stability of Drosophila alcohol dehydrogenase is presented. The potential of this technique for examining posttranslational control of in vivo enzyme concentrations is discussed.This work was supported by NSF grant #DEB 7815466 to J.M.Journal Paper No. J-9977 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2272.     

beta-Endorphin: evidence for the existence of opioid and non-opioid binding components for the tritiated human hormone in NG108-15 cells

Human beta-endorphin (beta h-EP) binding on neuroblastoma X glioma hybrid NG108-15 cells using tritiated human beta endorphin (3H-beta h-EP) as a primary ligand was found to have a component which was not displacable with [D-Ser2 )-Leu-enkephalin-Thr6 (DSLET). The beta h-EP binding on these cells after saturation of the delta opiate sites with 200 nM DSLET was further characterized with synthetic beta h-EP analogs. The nonopioid binding site appears to recognize beta h-EP-(6-31), beta h-EP-(21-31) and beta h-EP-(28-31). Under these conditions, these COOH-terminal segments fully displace the tritiated beta h-EP. However, beta h-EP-(1-27) does not further displace 3H-beta h-EP in the presence of DSLET. The fact that a combination of DSLET and beta h-EP-(6-31) results in a full displacement of 3H-beta h-EP provides direct evidence for the existence of two binding sites for beta h-EP in NG108-15 cells, one recognizing the NH2-terminal enkephalin sequence and the other the non-opioid COOH-terminal segment.     

Establishment of a dose-response relationship for reverse mutation at the HPRT (hypoxanthine guanine phosphoribosyl transferase) locus in L5178Y mouse lymphoma cells

L5178 mouse lymphoma cells were treated with the mismatching agent 6-hydroxy-aminopurine (HAP), a base analogue known to produce forward and reverse mutations in bacteria. Mutants with the phenotype deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT) were selected in 6-thioguanine (TG)-containing medium and isolated. Reverse mutations to Hhe HPRT-proficient phenotype oc occuredd both spontaneously and after treatment with ethyl nitrosourea (ENU), which suggested that the initial HAP treatment had induced point mutations at the HPRT locus.

Reconstruction experiments, in which a small number of wild-type cells together with different numbers of mutant cells were seeded in HAT medium, indicated that densities up to 10⁶ cells per ml can be used for the selection of revertants. Optimal expression of induced revertants was obtained two days after treatment.

The dose-response relationship for induction of reverse mutations by ENU appears not to deviate from linearity. The highest revertant frequency observed was 3.3 × 10⁻⁵ at an ENU concentration of 1 mM. The spontaneous reversion frequency per generation — based on 3 spontaneous revertants — was estimated to be 1.3 × 10⁻⁹. All revertants were indistinguishable from the parental wild-type line with respect to the activity as well as the electrophoretic mobility of HPRT.     

Heterologous expression of geraniol dehydrogenase for identifying the metabolic pathways involved in the biotransformation of citral by Acinetobacter sp. Tol 5

ABSTRACT

The biotransformation of citral, an industrially important monoterpenoid, has been extensively studied using many microbial biocatalysts. However, the metabolic pathways involved in its biotransformation are still unclear, because citral is a mixture of the trans-isomer geranial and the cis-isomer neral. Here, we applied the heterologous expression of geoA, a gene encoding geraniol dehydrogenase that specifically converts geraniol to geranial and nerol to neral, to identify the metabolic pathways involved in the biotransformation of citral. Acinetobacter sp. Tol 5 was employed in order to demonstrate the utility of this methodology. Tol 5 transformed citral to (1R,3R,4R)-1-methyl-4-(1-methylethenyl)-1,3-cyclohexanediol and geranic acid. Biotransformation of citral precursors (geraniol and nerol) by Tol 5 transformant cells expressing geoA revealed that these compounds were transformed specifically from geranial. Our methodology is expected to facilitate a better understanding of the metabolic pathways involved in the biotransformation of substrates that are unstable and include geometric isomers.     

CRK protein binds to two guanine nucleotide-releasing proteins for the Ras family and modulates nerve growth factor-induced activation of Ras in PC12 cells.

It has been reported that growth factors activate Ras through a complex of an adaptor type SH2-containing molecule, Grb2, and a Ras guanine nucleotide-releasing protein (GNRP), mSos. We report on the involvement of another adaptor molecule, CRK, in the activation of Ras. Overexpression of wild-type CRK proteins CRK-I and CRK-II enhanced the nerve growth factor (NGF)-induced activation of Ras in PC12 cells, although the basal level of GTP-bound active Ras was not altered. In contrast, mutants with a single amino acid substitution in either the SH2 or SH3 domain of the CRK-I protein inhibited the NGF-induced activation of Ras. Two GNRPs for the Ras family, mSos and C3G, were coimmunoprecipitated with the endogenous Crk proteins in PC12 cells. The association between C3G and the CRK mutants was dependent upon the presence of intact SH3. The SH2 domain of CRK bound to the SHC protein phosphorylated on tyrosine residues by NGF stimulation. The results demonstrate that, in addition to Grb2, CRK participates in signaling from the NGF receptor and that two GNRPs appear to transmit signals from these adaptor molecules to Ras.     

The use of lactate dehydrogenase (LDH) release kinetics for the evaluation of death and growth of mammalian cells in perfusion reactors

A general methodology is proposed to estimate the actual specific growth and death rate of mammalian cells in continuous perfusion reactors from the monitoring of the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in the culture medium. The procedure is illustrated on a perfusion culture of human tumor kidney cells growing on microcarriers and producing prourokinase (PUK). The intracellular LDH content of living attached cells is checked to be constant during the culture. However, cells detached from the microcarriers, and counted dead because of the uptake of trypan blue, have only released part of their intracellular LDH. In the culture medium, LDH is relatively stable as the loss of activity does not exceed 5% per day. The time variation of the LDH concentration in the medium is used to calculate the total amount of lysed and actually produced cells in the reactors, hence, the actual specific rates of cell growth and death. It is thus found that the stationary phase observed after 400 h of perfusion culture is the result of equal growth and death rates, with a daily renewal of living cells on the microcarriers near 10%. Moreover, for the cell line tested, the production of PUK is associated with cellular growth.