Biomedical subjects

M E Milla

Publications and source records attributed to M E Milla.

16 recordsLinked to original sources

PubMed2001

Neuronal localization of the TNFalpha converting enzyme (TACE) in brain tissue and its correlation to amyloid plaques.

The tumor necrosis factor (TNF)-alpha converting enzyme (TACE) can cleave the cell-surface ectodomain of the amyloid-beta precursor protein (APP), thus decreasing the generation of amyloid-beta (Abeta) by cultured non-neuronal cells. While the amyloidogenic processing of APP in neurons is linked to the pathogenesis of Alzheimer's disease (AD), the expression of TACE in neurons has not yet been examined. Thus, we assessed TACE expression in a series of neuronal and non-neuronal cell types by Western blots. We found that TACE was present in neurons and was only faintly detectable in lysates of astrocytes, oligodendrocytes, and microglial cells. Immunohistochemical analysis was used to determine the cellular localization of TACE in the human brain, and its expression was detected in distinct neuronal populations, including pyramidal neurons of the cerebral cortex and granular cell layer neurons in the hippocampus. Very low levels of TACE were seen in the cerebellum, with Purkinje cells at the granular-molecular boundary staining faintly. Because TACE was localized predominantly in areas of the brain that are affected by amyloid plaques in AD, we examined its expression in a series of AD brains. We found that AD and control brains showed similar levels of TACE staining, as well as similar patterns of TACE expression. By double labeling for Abeta plaques and TACE, we found that TACE-positive neurons often colocalized with amyloid plaques in AD brains. These observations support a neuronal role for TACE and suggest a mechanism for its involvement in AD pathogenesis as an antagonist of Abeta formation.

ADAM Proteins↗

PubMed2000

Protein kinase C-dependent alpha-secretase competes with beta-secretase for cleavage of amyloid-beta precursor protein in the trans-golgi network.

The release of amyloidogenic amyloid-beta peptide (Abeta) from amyloid-beta precursor protein (APP) requires cleavage by beta- and gamma-secretases. In contrast, alpha-secretase cleaves APP within the Abeta sequence and precludes amyloidogenesis. Regulated and unregulated alpha-secretase activities have been reported, and the fraction of cellular alpha-secretase activity regulated by protein kinase C (PKC) has been attributed to the ADAM (a disintegrin and metalloprotease) family members TACE and ADAM-10. Although unregulated alpha-secretase cleavage of APP has been shown to occur at the cell surface, we sought to identify the intracellular site of PKC-regulated alpha-secretase APP cleavage. To accomplish this, we measured levels of secreted ectodomains and C-terminal fragments of APP generated by alpha-secretase (sAPPalpha) (C83) versus beta-secretase (sAPPbeta) (C99) and secreted Abeta in cultured cells treated with PKC and inhibitors of TACE/ADAM-10. We found that PKC stimulation increased sAPPalpha but decreased sAPPbeta levels by altering the competition between alpha- versus beta-secretase for APP within the same organelle rather than by perturbing APP trafficking. Moreover, data implicating the trans-Golgi network (TGN) as a major site for beta-secretase activity prompted us to hypothesize that PKC-regulated alpha-secretase(s) also reside in this organelle. To test this hypothesis, we performed studies demonstrating proteolytically mature TACE intracellularly, and we also showed that regulated alpha-secretase APP cleavage occurs in the TGN using an APP mutant construct targeted specifically to the TGN. By detecting regulated alpha-secretase APP cleavage in the TGN by TACE/ADAM-10, we reveal ADAM activity in a novel location. Finally, the competition between TACE/ADAM-10 and beta-secretase for intracellular APP cleavage may represent a novel target for the discovery of new therapeutic agents to treat Alzheimer's disease.

Amyloid Precursor Protein Secretases↗

PubMed1999

Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.

ADAM Proteins↗

PubMed1997

Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.

Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.

ADAM Proteins↗

PubMed1996

Association of lipid A disaccharide synthase with aerobic glycerol-3-phosphate dehydrogenase in extracts of Escherichia coli.

Variants of the Escherichia coli UDP-GlcNAc O-acyltransferase (LpxA) and of the lipid A disaccharide synthase (LpxB) containing affinity chromatography tags (C-terminal histidine 8 [H8] tails) were constructed in order to investigate whether or not these enzymes interact with other E. coli proteins. These variants (LpxA-H8 and LpxB-H8) had specific activities in vitro that were similar to wild-type enzymes. Crude extracts made from E. coli cells expressing LpxA-H8 or LpxB-H8 were chromatographed over Ni(2+)-NTA-Agarose, and proteins purifying with the tagged proteins were identified by SDS-PAGE, followed by blotting and N-terminal microsequencing. At high levels of LpxB-H8 expression, two heat-shock proteins (DnaK and GroEL) were associated with the disaccharide synthase, but not with the acyltransferase. Another major protein recovered with LpxB-H8 (both at low and high levels of expression) was the aerobic glycerol-3-phosphate dehydrogenase (GlpD). The latter interaction was specific, since GlpD did not bind the affinity resin when the affinity tag was present on the UDP-GlcNAc O-acyltransferase (LpxA-H8). Velocity centrifugation experiments indicated that both wild-type LpxB and GlpD sedimented together under some conditions, but these aggregates were smaller than and distinct from inner membranes. Our findings suggest a possible new mechanism by which the biosynthetic pathways for lipid A and glycerophospholipids may be coordinated.

Acyltransferases↗

PubMed1996

Sequence determinants of folding and stability for the P22 Arc repressor dimer.

The Arc repressor is a small, homodimeric protein. Studies of mutant proteins show that the side chains that form the hydrophobic core are the most important determinants of structure. A variety of hydrogen bonds and salt bridges also contribute to stabilization of the native structure, but these can often be replaced by hydrophobic interactions. The transition state for folding/unfolding is dimeric and contains a large amount of buried hydrophobic surface, but the beta-sheet of native Arc is not formed. Moreover, relatively little side chain information appears to be used in the transition state, suggesting that tight packing of the hydrophobic core and optimization of hydrogen-bond geometry are events that occur later in folding.

Amino Acid Sequence↗

PubMed1995

P22 Arc repressor: transition state properties inferred from mutational effects on the rates of protein unfolding and refolding.

The kinetics of unfolding and refolding have been measured for a set of Arc repressor mutants bearing single amino acid substitutions at 44 of the 53 residue positions. Roughly half of the mutations cause significant changes in the unfolding and/or refolding rate constants. These substitutions alter the hydrophobic core, tertiary hydrogen bonds and salt bridges, and glycines with restricted backbone conformations. Overall, the mutations cause larger changes in the unfolding rates than the refolding rates, indicating that significantly less side-chain information is used between the denatured state and transition state than between the transition state and native state. The set of mutants displays reasonable Brønsted behavior, suggesting that many native interactions are partially formed in the transition state. Taken together, these observations suggest that the overall structure of most of the protein must be somewhat native-like in the transition state but without close, complementary packing of the hydrophobic core or good hydrogen bond geometry. Such a transition state is inconsistent with a model in which monomers fold to their correct conformations and then dock to form the dimer but supports a model in which folding and dimerization are concurrent processes.

Bacteriophage P22↗

PubMed1995

Critical side-chain interactions at a subunit interface in the Arc repressor dimer.

In the Arc repressor dimer, the side chains of Ile37 and Val41 in alpha-helix B pack against each other and against the symmetry-related side chains of Ile37' and Val41' in alpha-helix B' to form part of the hydrophobic core and the dimer interface. Following combinatorial mutagenesis of these positions, only the wild-type combination of hydrophobic residues was recovered as a fully active protein, and only a few conservative replacements were recovered as stably folded or partially active proteins. Equilibrium and kinetic studies of the folding of purified mutants show that the delta-CH3 groups of Ile37 and Ile37' contribute approximately 2 kcal/mol of dimer to protein stability and are involved in interactions that are only partially formed in the transition state for protein folding. Alanine substitution at either position 37 or 41 results in proteins which differ from wild type in being monomeric at a concentration of 10 microM, having reduced secondary structure, having solvent-exposed tryptophans, and showing non-cooperative thermal and urea denaturation transitions. These mutants appear to exist in a physiologically denatured state that is similar in many ways to the molten globule state.

Alanine↗

PubMed1995

Crystal structure, folding, and operator binding of the hyperstable Arc repressor mutant PL8.

Arc repressor is a small, dimeric DNA-binding protein that belongs to the ribbon-helix-helix family of transcription factors. Replacing Pro8 at the N-terminal end of the beta-sheet with leucine increases the stability of the mutant protein by 2.5 kcal/mol of dimer. However, this enhanced stability is achieved at the expense of significantly reduced DNA binding affinity. The structure of the PL8 mutant dimer has been determined to 2.4-A resolution by X-ray crystallography. The overall structure of the mutant is very similar to wild type, but Leu8 makes an additional interstrand hydrogen bond at each end of the beta-sheet of the mutant, increasing the total number of beta-sheet hydrogen bonds from six to eight. Comparison of the refolding and unfolding kinetics of the PL8 mutant and wild-type Arc shows that the enhanced stability of the mutant is accounted for by a decrease in the rate of protein unfolding, suggesting that the mutation acts to stabilize the native state and that the beta-sheet forms after the rate-limiting step in folding. The reduced operator affinity of the PL8 dimer appears to arise because the mutant cannot make the new interstrand hydrogen bonds and simultaneously make the wild-type set of contacts with operator DNA.

Crystallography, X-Ray↗

PubMed1994

Purification of the Golgi adenosine 3'-phosphate 5'-phosphosulfate transporter, a homodimer within the membrane.

Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen of the Golgi apparatus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the sulfate donor in these reactions, must be transported from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus. We have identified and purified to apparent homogeneity the rat liver Golgi membrane PAPS transporter by a combination of conventional and affinity chromatography as well as photoaffinity radiolabeling with adenosine 3',5'-bisphosphate, a competitive inhibitor of PAPS transport. The transporter, a 75-kDa protein, was purified 70,000-fold over homogenate (6% yield) and transported PAPS into phosphatidylcholine liposomes selectively and in a saturable manner (apparent Km of 1.7 microM). Radiation target-inactivation analyses of the transport activity in rat liver Golgi vesicles, together with the above described biochemical approaches, demonstrate that the PAPS transporter within the Golgi membrane is a homodimer.

Affinity Labels↗

PubMed1994

P22 Arc repressor: folding kinetics of a single-domain, dimeric protein.

The rate constants for refolding and unfolding of the P22 Arc repressor dimer have been determined by stop-flow fluorescence experiments. Under most conditions, refolding is described well as a two-state reaction with a bimolecular rate-limiting step (kf approximately 10(7) M-1 s-1). A unimolecular step appears to become co-rate limiting at high protein concentrations. The urea dependence of the refolding reaction suggests that about 75% of the total burial of hydrophobic surface occurs between the unfolded state and the transition state for folding. Hydrophobic interactions are also evidenced by the temperature dependence of the refolding reaction; the rate increases with temperature and Arrhenius plots are curved, as expected for a reaction that proceeds with a significant heat capacity change. The refolding of Arc also proceeds more rapidly as the salt concentration is raised, presumably because repulsive interactions between monomers are screened. At a protein concentration of 10 microM, the apparent rate constant for refolding of the Arc dimer is approximately 100 s-1, as fast as the refolding of many monomeric proteins. The rate constant for unfolding is approximately 0.1 s-1, corresponding to a half-life of less than 10 s for the folded Arc dimer. This rate of unfolding is very fast in comparison to that of other characterized proteins and implies that a free Arc molecule must unfold and refold hundreds of times per generation in the cell.

DNA-Binding Proteins↗

PubMed1994

Scanning mutagenesis of the Arc repressor as a functional probe of operator recognition.

Protein-DNA and protein-protein interactions are central to most biological regulation, and yet our understanding of these macromolecular recognition events is still incomplete. Both types of interactions are critical for the function of the Arc repressor. The functional importance of residues in or near its operator DNA-binding surface and dimer-dimer interaction surface has been probed by alanine-scanning mutagenesis. Mutations in three categories cause large binding defects: beta-sheet side chains that directly interact with DNA bases; side chains that link different DNA-binding regions of Arc, and side chains required to maintain the active DNA-binding conformation.

Bacteriophage P22↗

PubMed1994

Protein stability effects of a complete set of alanine substitutions in Arc repressor.

The equilibrium stabilities of a complete set of single alanine-substitution mutants of the Arc repressor of bacteriophage P22 have been determined by thermal and urea denaturation experiments. Only half the alanine substitutions cause significant changes in stability, with the most deleterious mutations affecting side chains in the hydrophobic core or in salt bridges and hydrogen bonds which are protected from solvent. The five mutations that are most destabilizing affect a cluster of core residues that seem to form a structural foundation for Arc.

Alanine↗

PubMed1993

P22 Arc repressor: enhanced expression of unstable mutants by addition of polar C-terminal sequences.

Many mutant variants of the P22 Arc repressor are subject to intracellular proteolysis in Escherichia coli, which precludes their expression at levels sufficient for purification and subsequent biochemical characterization. Here we examine the effects of several different C-terminal extension sequences on the expression and activity of a set of Arc mutants. We show that two tail sequences, KNQHE (st5) and H6KNQHE (st11), increase the expression levels of most mutants from 10- to 20-fold and, in some cases, result in restoration of biological activity in the cell. A third tail sequence, HHHHHH (st6), was not as effective in increasing mutant expression levels. All three tail sequences are functionally and structurally silent, as judged by their lack of effects on the DNA binding activity and stability of otherwise wild-type Arc. The properties of the st11 tail sequence make it an efficient system for the expression and purification of mutant Arc proteins, both because mutant expression levels are increased and because the proteins can be rapidly purified using nickel-chelate affinity chromatography. Arc mutants containing the EA28, RL31, and SA32 mutations were purified in the st11 background. The thermodynamic stability of the EA28 mutant (delta delta Gu approximately -0.4 kcal/mol) is reduced modestly compared to the st11 parent, whereas the RL31 mutant (delta delta Gu approximately -3.0 kcal/mol) and SA32 mutant (delta delta Gu approximately -3.3 kcal/mol) are substantially less stable.

Amino Acid Sequence↗

PubMed1992

Reconstitution into proteoliposomes and partial purification of the Golgi apparatus membrane UDP-galactose, UDP-xylose, and UDP-glucuronic acid transport activities.

Previous studies in vitro on proteoglycan biosynthesis from our laboratory have shown that nucleotide sugar precursors of all the sugars of the linkage oligosaccharides (xylose, galactose, and glucuronic acid) and of the glycosaminoglycans (N-acetylglucosamine, N-galactosamine, and glucuronic acid) are transported by specific carriers into the lumen of Golgi vesicles. More recently, we also reported the reconstitution in phosphatidylcholine liposomes of detergent-solubilized Golgi membrane proteins containing transport activities of CMP-sialic acid and adenosine-3'-phosphate-5'-phosphosulfate. We have now completed the successful reconstitution into liposomes of the Golgi membrane transport activities of UDP-galactose, UDP-xylose, and UDP-glucuronic acid. Transport of these nucleotide sugars into Golgi protein proteoliposomes occurred with the same affinity, temperature dependence, and sensitivity to inhibitors as observed with intact Golgi vesicles. Preloading of proteoliposomes with UMP, the putative antiporter for Golgi vesicle transport of these nucleotide sugars, stimulated transport of the nucleotide sugars by 2-3-fold. Transport of UDP-xylose into Golgi protein proteoliposomes was dependent on the presence of endogenous Golgi membrane lipids while that of UDP-galactose and UDP-glucuronic acid was not. This suggests a possible stabilizing or regulatory role for Golgi lipids on the UDP-xylose translocator. Finally, we have also shown that detergent-solubilized Golgi membrane translocator proteins can be partially purified by an ion-exchange chromatographic step before successful reconstitution into liposomes, demonstrating that this reconstitution approach can be used for the biochemical purification of these transporters.

Animals↗

PubMed1989

Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes.

We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide sulfate into their lumen. These transport activities are organelle and substrate specific and are characterized by apparent Km for nucleotide derivatives in the low micromolar range. As part of our goal of purifying and characterizing the above transport proteins, we have reconstituted a protein extract from rat liver Golgi membranes into phosphatidylcholine liposomes. The resulting proteoliposomes transport CMP-N-acetylneuraminic acid (CMP-AcNeu) and adenosine 3'-phosphate 5'-phosphosulfate with very similar affinity and inhibition characteristics as intact Golgi vesicles. Sialic acid and sodium sulfate, which are transported only very slowly into the lumen of Golgi vesicles, are transported at low rates by the reconstituted proteoliposomes. Neither rough endoplasmic reticulum-derived vesicles nor proteoliposomes made from proteins of the rough endoplasmic reticulum transport CMP-AcNeu. The above results demonstrate that this reconstituted system can be used for further purification and characterization of nucleotide sugar and nucleotide sulfate translocator proteins. This approach should also be useful to study membrane transport proteins of lysosomes and endosomes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗