Availability and canonical positioning of key amino acids of ornithine-decarboxylase degron is insufficient for alpha-fetoprotein degradation

Ornithine decarboxylase (ODC) degrades in proteasome in a ubiquitin-independent manner with the half life of approximately 2 h. Thirty seven C-terminal amino acids of this enzyme constitute a fragment known as the degradation signal (degron), which is responsible for the effectiveness of protein degradation. Among these amino acids, the key positions have recently been mapped (Cys441 and Ala442). Mutations of the key amino acids led to ODC general stabilization, whereas substitution of other amino acids had no significant influence on the ODC degron activity. In addition, deletions or insertions into the region located between the key amino acids and ODC C-end diminished significantly the rate of protein degradation; hence, the distance (remoteness) of these amino acids from ODC C-end is, probably, of crucial importance. Taking into account these data, we have introduced the key amino acids that determine ODC-degron activity into alpha-fetoprotein with the truncated export signal (ΔAFP) so that their positioning was 20 amino-acid away from the C-end (ΔAFPCAG and ΔAFPLCAG). Secretion of ΔAFP and the modified proteins from cells was impossible because of a removal of the N-terminal export signal. Computer analysis of ΔAFP and the derivative ΔAFPCAG and ΔAFPLCAG revealed no significant changes in protein hydrophobicity or in the secondary structure of C-terminal region. The in vitro experiments on HEK293T cells using MG132 proteasome inhibitor and translation inhibitor cycloheximide have demonstrated similar stability of ΔAFP and the derivative ΔAFPCAG and ΔAFPLCAG in cells. Thus, introduction of the key amino acids of ODC degron at the key positions relative to the C-end of ΔAFP did not change the parameters of protein degradation. Perhaps, some other still unknown amino acids are important for ODC-degron functioning. It may well be that ΔAFP conformation prevents interaction of the protein C-end with proteasome.     

Recognition of C-terminal amino acids in tubulin by pore loops in Spastin is important for microtubule severing

Spastin, an AAA ATPase mutated in the neurodegenerative disease hereditary spastic paraplegia, severs microtubules. Many other AAA proteins form ring-shaped hexamers and contain pore loops, which project into the ring's central cavity and act as ratchets that pull on target proteins, leading, in some cases, to conformational changes. We show that Spastin assembles into a hexamer and that loops within the central pore recognize C-terminal amino acids of tubulin. Key pore loop amino acids are required for severing, including one altered by a disease-associated mutation. We also show that Spastin contains a second microtubule binding domain that makes a distinct interaction with microtubules and is required for severing. Given that Spastin engages the MT in two places and that both interactions are required for severing, we propose that severing occurs by forces exerted on the C-terminal tail of tubulin, which results in a conformational change in tubulin, which releases it from the polymer.     

S-adenosyl-L-methionine decarboxylase during lymphocyte transformation: decreased degradation in the presence of a specific inhibitor

S-Adenosyl-L-methionine decarboxylase (AmDC) activity increased 20- to 25-fold in a biphasic manner during Concanavalin A-induced lymphocyte transformation. When a potent inhibitor of this enzyme, methylglyoxal bis (guanylhydrazone), was added to transforming cultures, AmDC rapidly increased beyond the fully induced level and eventually reached a specific activity 2500 times that found in non-transformed lymphocytes. Measurements of the decline in enzyme activity in the presence of cycloheximide indicated that the half-life increased from 40 minutes to at least 20 hours during inhibitor treatment. It is likely that this change in the rate of AmDC degradation was primarily, if not solely, responsible for the large increases observed.     

Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids

Amino Acids - Neurotensin (NT) (pGlu–Leu–Tyr–Glu–Asn–Lys–Pro–Arg–Arg–Pro–Tyr–Ile–Leu) exerts a dual function as a...     

Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids

Intracellular degradation of exogenous (serum) proteins provides a source of amino acids for cellular protein synthesis. Pinocytosis serves as the mechanism for delivering exogenous protein to the lysosomes, the major site of intracellular degradation of exogenous protein. To determine whether the availability of extracellular free amino acids altered pinocytic function, we incubated monolayers of pulmonary alveolar macrophages with the fluid-phase marker, [14C]sucrose, and we dissected the pinocytic process by kinetic analysis. Additionally, intracellular degradation of endogenous and exogenous protein was monitored by measuring phenylalanine released from the cell monolayers in the presence of cycloheximide. Results revealed that in response to a subphysiological level of essential amino acids or to amino acid deprivation, (a) the rate of fluid-phase pinocytosis increased in such a manner as to preferentially increase both delivery to and size of an intracellular compartment believed to be the lysosomes, (b) the degradation of exogenously supplied albumin increased, and (c) the fraction of phenylalanine derived from degradation of exogenous albumin and reutilized for de novo protein synthesis increased. Thus, modulation of the pinosome-lysosome pathway may represent a homeostatic mechanism sensitive to the availability of extracellular free amino acids.     

The cyclin-dependent kinase inhibitory domain of the yeast Sic1 protein is contained within the C-terminal 70 amino acids

By inhibiting the activity of Cdc28/Clb cyclin-dependent protein kinase (CDK) complexes, Sic1 prevents the premature initiation of S phase in the yeast Saccharomyces cerevisiae. By testing a series of Sic1 truncation mutants, we have mapped the minimal domain necessary for Cdc28/Clb inhibition in vivo to the C-terminal 70 amino acids of Sic1. Site-directed mutagenesis was used to show that a sequence that matches the zRxL motif found in mammalian CDK inhibitors is essential for Sic1 function. This motif is not found in the Schizosaccharomyces CDK inhibitor p25^rum1, which appears to be a structural and functional homolog of Sic1. Based on the mutational data and sequence comparisons, we argue that Sic1 and p25^rum1 are structurally distinct from the known mammalian CDK inhibitors, but may bind CDK complexes in a manner more closely resembling CDK substrates like the retinoblastoma and E2F proteins. Received: 3 February 1999 / Accepted: 23 April 1999     

Stimulation of glutathione degradation by amino acids: lack of stereospecificity

The rate of degradation of glutathione by rat kidney slices has been analysed. In the absence of exogenous amino acids a half-life of 84 min is found. In the presence of the L-isomer of three amino acids which are good substrates for gamma-glutamyl transpeptidase the rate of degradation is increased in a concentration-dependent manner. The stimulatory effect is not stereospecific, the D-isomers having a similar effect to their L-enantiomers. These findings indicate that perturbations in glutathione metabolism need not be due to the stimulation of active transport mediated by gamma-glutamyl transpeptidase.     

Photooxidation of amino acids in the presence of methylene blue

A systematic study was made of the photochemical action of methylene blue on amino acids.Tyrosine, tryptophan, histidine, methionine, and cystine were highly reactive during the photoöxidation; the rest of the amino acids acted sluggishly or not at all.In tyrosine, tryptophan, and histidine, the entire oxygen uptake and CO₂ evolution were due to the cyclic nucleus, involving rupture of the rings.During the photochemical action of methylene blue on tyrosine, tryptophan, and methionine, intermediary oxidizing agents were formed; in methionine this was shown to be H₂O₂.The photoöxidation of methionine resulted in the formation of methionine sulfoxide as an end product.Iodometric titration and measurement of ultraviolet absorption during irradiation of methionine indicate the formation of an intermediary dehydrogenation product which appears to differ from Lavine's dehydromethionine.Cystine was photoöxidized, probably beyond the cysteic acid stage.Peptide bonds did not participate in the photochemical action of methylene blue.Methylation of the α-amino group of lysine to the corresponding secondary and tertiary compounds produced increased reactivity in the photoöxidation.     

A mutant streptokinase lacking the C-terminal 42 amino acids is less reactive with preexisting antibodies in patient sera

Streptokinase (SK) is an efficacious thrombolytic drug for the treatment of myocardial infarction. Because of its immunogenicity, patients receiving SK therapy develop high anti-SK antibody (Ab) titers, which might provoke severe allergic reactions and neutralize SK activity. In this report we studied the reactivity of a synthetic 42-residue peptide resembling SKC-2 C-terminus with patient sera. SKC-2(373-414) peptide was recognized by 39 and 64% of patients, before and after SKC-2 therapy, respectively. An SKC-2 deletion mutant (mut-C42), lacking the same 42 C-terminal residues, was constructed and expressed in Escherichia coli. Recognition of mut-C42 by preexisting Abs from patient sera was 51 and 68% of reactivity to SKC-2, as assessed by direct binding and competition assays, respectively. For most of the patients, mut-C42-neutralizing activity titer (NAT) significantly decreased with respect to SKC-2-NAT. This study opens the possibility of producing a less immunogenic variant of SK, which could constitute a preferred alternative for thrombolytic therapy.