The magnesium protein interaction in nucleotide complexes of phos-phoglycerate kinase

The X-ray structure determination of yeast phosphoglycerate kinase and subsequent substrate binding studies have helped to define the binding sites for the triose and nucleoside phosphate substrates. This communication deals with one feature of the binding site—the location of an aspartic acid residue close to the phosphoryl binding site of the nucleotide substrate—and relates this and other structural features of the active site to the properties of this enzyme as deduced from nuclear magnetic resonance studies.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Activity and specificity of human aldolases

The structure of the type I fructose 1,6-bisphosphate aldolase from human muscle has been extended from 3 A to 2 A resolution. The improvement in the resulting electron density map is such that the 20 or so C-terminal residues, known to be associated with activity and isozyme specificity, have been located. The side-chain of the Schiff's base-forming lysine 229 is located towards the centre of an eight-stranded beta-barrel type structure. The C-terminal "tail" extends from the rim of the beta-barrel towards lysine 229, thus forming part of the active site of the enzyme. This structural arrangement appears to explain the difference in activity and specificity of the three tissue-specific human aldolases and helps with our understanding of the type I aldolase reaction mechanism.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Site-selective glycosylation of proteins: creating synthetic glycoproteins

In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs). Glycosylation is by far the most diverse of the PTM processes. Natural protein production methods typically produce PTM or glycoform mixtures within which function is difficult to dissect or control. Chemical tagging methods allow the precise attachment of multiple glycosylation modifications to bacterially expressed (bare) protein scaffolds, allowing reconstitution of functionally effective mimics of glycoproteins in higher organisms. In this way combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein-PTM interactions. This protocol describes the modification of Cys residues in proteins using glycomethanethiosulfonates and glycoselenenylsulfides and the modification of azidohomoalanine residues, introduced by Met replacement using auxotrophic Met(-) Escherichia coli strains, with glycoalkynes and the combination of these techniques for the creation of dual-tagged proteins. Each glycosylation procedure outlined in this protocol can be achieved in half a day.     

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology

Background

Neurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimer's disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimer's disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role.

Results

We have investigated the effects of tau phosphorylation by glycogen synthase kinase-3β (GSK-3β) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3β is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimer's disease brain.

Conclusion

These results suggest that phosphorylation of tau by GSK-3β promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.     

Tau polymerization: role of the amino terminus

The abnormal polymerization of the tau molecule into insoluble filaments is a seminal event in the neurodegenerative process underlying Alzheimer's disease. Previous experimentation has shown that the microtubule-binding repeat region of the molecule is vital for its ability to polymerize in vitro into filaments similar to those found in Alzheimer's disease. However, it is becoming clear that regions outside the microtubule-binding repeat, such as exons 2 and 3 and the carboxy-terminal tail, can greatly influence its polymerization. Since it has been previously postulated that the amino terminus of tau could be involved in generating pathological conformations in the disease state, its role in the polymerization process was investigated. This report demonstrates that the removal of the amino terminus greatly inhibits the polymerization of the tau molecule, reducing both the rate and extent of polymerization. These results support the hypothesis that the ability of tau to form specific conformations involving the amino terminus is an early event in the formation of tau polymers in the disease state. Furthermore, the mutation of arginine 5 to leucine ((R)5(L)), mimicking an amino-terminal tau mutation found in a single case of FTDP-17, enhances the polymerization of the tau molecule. Therefore, the amino terminus of the tau molecule, while largely overlooked in studies of its polymerization, is a significant contributor to the polymerization process.