Lucía Collados

Human placental atp-diphosphohydrolase: Biochemical characterization, regulation and function

1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident M(r) and pI values of both ATPase-ADPase activities. 2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher. 3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DES and slightly by DCCD. 4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A. 5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of -SH, COO-, -OH, and probably also Tyr and Trp. 6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes. 7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.

Inhibition of cyclin-dependent kinase 5 but not of glycogen synthase kinase 3-β prevents neurite retraction and tau hyperphosphorylation caused by secretable products of human T-cell leukemia virus type I-infected lymphocytes

Human T‐cell leukemia virus type I (HTLV‐I)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T181 in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH‐SY5Y cells incubated with supernatant from MT‐2 cells (HTLV‐I‐infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T181 is attributable to glycogen synthase kinase 3‐β (GSK3‐β) and cyclin‐dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH‐SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3‐β and CDK5 in this process by using specific kinase inhibitors (LiCl, TDZD‐8, and roscovitine). Changes in both GSK3‐β active and inactive forms were followed by measuring the regulatory phosphorylable sites (S9 and Y216, inactivating and activating phosphorylation, respectively) together with changes in β‐catenin protein levels. Our results showed that LiCl and TDZD‐8 were unable to prevent MT‐2 supernatant‐mediated neurite retraction and also that neither Y216 nor S9 phosphorylations were changed in GSK3‐β. Thus, GSK3‐β seems not to play a role in T181 hyperphosphorylation. On the other hand, the CDK5 involvement in tau phosphorylation was confirmed by both the increase in its enzymatic activity and the absence of MT‐2 neurite retraction in the presence of roscovitine or CDK5 siRNA transfection.

TIMPs and MMPs expression in CSF from patients with TSP/HAM

The tropical spastic paraparesis or human T-cell lymphotropic virus associated myelopathy (TSP/HAM), has been related with an overexpression of matrix metalloproteinases (MMPs), especially MMP-9. Initial studies of reverse zymography with cerebrospinal fluid (CSF) from TSP/HAM patients, and controls showed the presence of TIMPs, endogenous MMP inhibitors. We determined in CSF the levels of TIMPs by immunoanalysis in 25 patients with TSP/HAM, and compared with two groups: controls and patients with acute and subacute inflammatory neurological diseases. We found that TIMP-2, TIMP-3 and TIMP-4 levels were significantly higher than in controls in both TSP/HAM and inflammatory patients, while TIMP-1 was increased only in the inflammatory group. Levels of MMP-3 and MMP-9 from the two groups of patients showed a significant upregulation in CSF. In the CSF of around the 70% of TSP-HAM and inflammatory patients the presence MMP-9 was detected by zymography, but not in controls. MMP-2 was only overexpressed in the acute inflammatory group. The active form of MMP-2 was observed in both groups of patients with a similar high frequency (60%). MMPs overexpressions are independent of the evolution time of the disease in TSP/HAM. The chronic overexpression of these extracelullar matrix proteins detected in CSF of TSP/HAM should be indirectly produced by secreted viral proteins being responsible for the progression of this disease, accounting for the observed differences with acute inflammatory patients. Our results support the existence of an imbalance between MMPs and their endogenous tissue inhibitors, which could be a pathogenic factor in the chronicity of TSP/HAM.

Microtubule proteins and their post-translational forms in the cerebrospinal fluid of patients with paraparesis associated with HTLV-I infection and in SH-SY5Y cells: An in vitro model of HTLV-I-induced disease

HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by axonal degeneration of the corticospinal tracts. The specific requirements for transport of proteins and organelles to the distal part of the long axon are crucial in the corticospinal tracts. Microtubule dysfunction could be involved in this disease, configuring an axonal transport disease. We measured tubulin and its post-translational modified forms (acetylated and tyrosinated) in CSF of patients and controls, as well as tau and its phosphorylated forms. There were no significant differences in the contents of tubulin and acetyl-tubulin between patients and controls; tyrosyl-tubulin was not detected. In HAM/TSP, tau levels were significantly reduced, while the ratio of pT181/total tau was higher in patients than in controls, this being completely different from what is reported in other neurodegenerative diseases. Phosphorylation at T181 was also confirmed by Mass Spectrometry analysis. Western Blotting with monospecific polyclonal antibodies against pS199, pT205, pT231, pS262, pS356, pS396, pS404 and pS422 did not show differences in phosphorylation in these residues between patients and controls. Treating human SH-SY5Y neuroblastoma cells, a well-known in vitro neurite retraction model, with culture supernatant of MT-2 cells (HTLV-I infected cell line that secretes the viral Tax protein) we observed neurite retraction and an increase in tau phosphorylation at T181. A disruption of normal phosphorylation of tau protein in T181 could result in its dysfunction, contributing to axonal damage.

ATP-DIPHOSPHOPHYDROLASE ACTIVITY IN RAT HEART TISSUE

Extracellular nucleotides interact with specific receptors on the cell surface and are locally metabolized by ecto‐nucleotidases. Biochemical characterization of the ATPase and ADPase activities detected in rat heart sarcolemma, under conditions where mitochondrial ATPase and adenylate kinase were blocked, supports our proposal that both activities correspond to a single enzyme, known as ATP‐diphosphohydrolase or apyrase. The physiological function of this enzyme could be dephosphorylation of the nucleotides present in the interstitial heart compartment acting together with 5′‐nucleotidase. Both hydrolytic activities have similarities in: sarcolemma localization, bivalent metal ion dependence, optimum pH, effect of several amino acid residue modifiers, competitive inhibition of nucleotide analogs, and broad nucleoside di‐and triphosphate specificity. The ATPase activity could not be separated from the ADPase either through isoelectrofocusing or electrophoresis under acid conditions.

Changes in apyrase activity in uterus and mammary gland during the lactogenic cycle.

1. The purpose of this present research was to explore the possible roles of ATP-diphosphohydrolase (apyrase) in two tissues with high energetic demands during cell proliferation and differentiation. 2. Changes in apyrase activities during the pregnancy lactation cycle were examined in the rat uterus and mammary gland. 3. A significant decrease in apyrase activity (ATPase-ADPase) was observed in the pregnant uterus; this observation correlates with a minor inhibitory effect on platelet aggregation. 4. In mammary gland, the enzyme activity increases during lactation in parallel with an increase in blood supply, synthesis of glycoproteins and cell proliferation. 5. Apyrase activity did not change during the estrous cycle. Estradiol administration to rats slightly increased (20%) both ATPase-ADPase activities. 6. The probable function of apyrase is finally discussed, based on its substrate specificity and subcellular localization.

Comparative study of CSF neurofilaments in HTLV-1-associated myelopathy/tropical spastic paraparesis and other neurological disorders.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive CNS disease leading to corticospinal tract degeneration. Various degenerative diseases have increased neurofilament subunit concentration in cerebrospinal fluid (CSF), frequently showing hyperphosphorylation in neurofilaments. The aim of this study was to determine if there were elevated concentrations of neurofilament light subunit (NFL) and phosphorylated forms of neurofilament heavy subunit (PNFH) in HAM/TSP CSF. NF concentrations were compared with those of controls and patients with neurodegenerative diseases associated with other retroviruses (HIV-associated dementia, HAD) and a form of prion disease (familiar Creutzfeldt-Jakob, FCJD). Western blotting of CSF with antibodies against NFL showed two immunoreactive bands of 66 and 59 kDa, the latter probably corresponding to a partially degraded NFL form. The concentration of the 59-kDa form was not different in HAM/TSP compared with controls, but it was significantly increased in HAD and FCJD groups. ELISA assay for PNFH did not show differences among HAM/TSP, HAD, and control groups, while PNFH concentration was significantly elevated in FCJD. Our results show that CSF NFL and PNFH are not molecular markers of axonal damage for HAM/TSP probably due to the slow progression of this disease. NFL phosphorylation studies required previous immunoprecipitation from CSF for mass spectrometric analysis. This preliminary analysis indicated phosphorylation at S472 and at some other residues.

Characterization of ATP-diphosphohydrolase from rat mammary gland

ATP-diphosphohydrolase (or apyrase) hydrolyses nucleoside di- and triphosphates in the presence of millimolar concentration of divalent cations. It is insensitive towards sulfhydryl and aliphatic hydroxyl-selective reagents and to specific inhibitors of ATPases. We present further evidence that ATPase and ADPase activities present in rat mammary gland correspond to apyrase. Two kinetic approaches have been employed, competition plot and chemical modification with group-selective reagents. The M(r) of these activities was determined by 60Co radiation-inactivation. The kinetic approaches employed, competition plot (which discriminate whether competitive reactions occur at the same site) and chemical modification, point to the presence of a single protein which hydrolyses ATP and ADP. The similar M(r) values of ATPase and ADPase activities also support this proposal. ATPase and ADPase activities of mammary gland show a similar sensitivity or insensitivity towards several chemical modifiers. These results suggest that this enzyme is ATP-diphosphohydrolase, also known as apyrase. The results obtained are compared with the ones obtained by us and other authors with the enzyme isolated from other sources.

ECTO-ATP-Diphosphohydrolase from Normal and Abnormal Placenta

Vasoactive properties of adenosine and ATP on placenta has been described.1 Adenosine has been associated with vasodilation and ATP has a dual effect (vasodilation and vasoconstriction) depending on the receptors to which it binds. ADP is a powerful platelet stimulant, whereas ATP and adenosine inhibits ADP-induced platelet aggregation2. Mechanims of inactivation of circulating nucleotides, which imply the sequential degration of nucleoside di- and triphosphates to adenosine, are important in the control of their vascular actions. The scavenging of extracellular nucleotides is only possible in the dephosphorylated form3. We propose that ATP-diphosphohydrolase or apyrase (E. C. 3.6.1.5), characterized by its ATPase and ADPase activities, could be involved in the regulation of the extracellular nucleotide levels acting together with 5′-nueleotidase4. This enzyme has been found localized as an ectoenzyme in erythrocytes and platelets5,6, as well as in endothelial and smooth muscle cells7. Apyrase is different from the high affinity Ca2+-ATPase involved in the Ca2+ transport8 and its function has been associated to platelet aggregation inhibition. The placental tissue does not produce significant amounts of PGI2, a potent platelet antiaggregant and ADPase activity could be the most important part of the anti-platelet activity9.

ATP-Diphosphohydrolase Activity from Mammary Gland

ATP-diphosphohydrolase (EC 3.6.1.5), also known as apyrase, has been found in rat mammary gland and several other animal tissuesanimal tissues1,2,3,4. The main kinetic characteristics of this enzyme are the broad specificity towards nucleoside di- and triphosphates and the dependence of either Ca2+ or Mg2+. These characteristics differentiate apyrase from the Ca2+transport ATPase and the Golgi UDPase described in mammary gland1,4. The Ca2+ transport enzyme isolated from plasma membrane of mammary carcinoma cells requires Mg2+ in addition to micromolar concentrations of Ca2+, it is calmodulin dependent and has a Mr of 150 kDa by SDS/PAGE5. The UDPase activity6, considered a Golgi apparatus marker, is specific for UDP, GDP and IDP and the optimum pH (below 7.0) is lower than the optimum for apyrase from animal tissues1. We have proposed that the function of this plasma membrane enzyme in mammary gland might be related to the extracellular nucleotide metabolism7. The action of apyrase, which removes Pi from ATP and ADP, and 5′-nucleotidase which finally produces adenosine, could play a role in the scavenging of purine ring ensuring its reincorporation into the cell. Furthermore this enzyme might have an important function in the modulation of extracellular ATP-induced changes of intracellular calcium activity, mediating a P2-type purinergic receptor, recently described in mammary gland epithelial cells8. If these proposed functions are correct, apyrase location in the plasma membrane should be as an ecto-enzyme. It has been shown by Carraway et al.9 in mammary gland and its tumours the presence of an ecto-ATPase activity stimulated by either Mg2+ or Ca2+.