Biomedical subjects

A Ian Smith

Publications and source records attributed to A Ian Smith.

25 records · Page 2Linked to original sources

PubMed2003

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism.

The closely related metalloendopeptidases EC (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.

Animals↗

PubMed2003

Metalloendopeptidases EC 3.4.24.15/16 regulate bradykinin activity in the cerebral microvasculature.

Bradykinin is a vasoactive peptide that has been shown to increase the permeability of the cerebral microvasculature to blood-borne macromolecules. The two zinc metalloendopeptidases EC (EP 24.15) and EC (EP 24.16) degrade bradykinin in vitro and are highly expressed in the brain. However, the role that these enzymes play in bradykinin metabolism in vivo remains unclear. In the present study, we investigated the role of EP 24.15 and EP 24.16 in the regulation of bradykinin-induced alterations in microvascular permeability. Permeability of the cerebral microvasculature was assessed in anesthetized Sprague-Dawley rats by measuring the clearance of 70-kDa FITC dextran from the brain. Inhibition of EP 24.15 and EP 24.16 by the specific inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA-2) resulted in the potentiation of bradykinin-induced increases in cerebral microvessel permeability. The level of potentiation was comparable to that achieved by the inhibition of angiotensin-converting enzyme. These findings provide the first evidence of an in vivo role for EP 24.15/EP 24.16 in brain function, specifically in regulating alterations in microvessel permeability induced by exogenous bradykinin.

Animals↗

PubMed2003

Albumin fragments in normal rat urine are derived from rapidly degraded filtered albumin.

Filtered albumin is excreted as a heterogeneous population of albumin-derived molecules resulting from degradation during renal passage. In order to understand the dynamics of this degradation process, albumin clearance was studied over a short-term (minutes) and a long-term (7 days) by both radioactivity and radioimmunoassay. The radiolabelled material in the urine was also analysed extensively by using size exclusion chromatography, size selective filtration and high performance liquid chromatography. These studies demonstrate that during renal passage, albumin degradation to fragments in the size range of 500-10,000 occurs in a matter of minutes. The fragments are not detected by using radioimmunoassay. Steady state excretion rates or fractional clearance of radiolabelled albumin occur over a similar time period. Both rates of degradation and approach to steady-state clearance, while rapid, were considerably slower than the transit time for molecules in the Bowman's capsule and early tubular lumen. The results are consistent with an extremely rapid lysosomal uptake of filtered albumin, and degradation and regurgitation of the albumin-derived peptide fragments into the tubular lumen prior to excretion.

Albumins↗

PubMed2002

Inhibitors of metalloendopeptidase EC 3.4.24.15 and EC 3.4.24.16 stabilized against proteolysis by the incorporation of beta-amino acids.

The enzyme EC 3.4.24.15 (EP 24.15) is a zinc metalloendopeptidase whose precise function in vivo remains unknown but is thought to participate in the regulated metabolism of a number of specific neuropeptides. The lack of stable and selective inhibitors has hindered the determination of the exact function of EP 24.15. Of the limited number of EP 24.15 inhibitors that have been developed, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (CFP) is the most widely studied. CFP is a potent and specific inhibitor, but it is unstable in vivo due to cleavage between the alanine and tyrosine residues by the enzyme neprilysin (EP 24.11). This cleavage by EP 24.11 generates a potent inhibitor of angiotensin converting enzyme, thereby limiting the use of CFP for in vivo studies. To develop specific inhibitors of EP 24.15 that are resistant to in vitro and potentially in vivo proteolysis by EP 24.11, this study incorporated beta-amino acids replacing the Ala-Tyr scissile alpha-amino acids of CFP. Both C2 and C3 substituted beta-amino acids were synthesized and substituted at the EP 24.11 scissile Ala-Tyr bond. Significant EP 24.15 inhibitory activity was observed with some of the beta-amino acid containing analogues. Moreover, binding to EP 24.11 was eliminated, thus rendering all analogues containing beta-amino acids resistant to degradation by EP 24.11. Selective inhibition of either EP 24.15 or EP 24.16 was also observed with some analogues. The results demonstrated the use of beta-amino acids in the design of inhibitors of EP 24.15 and EP 24.16 with K(i)'s in the low micromolar range. At the same time, these analogues were resistant to cleavage by the related metalloendopeptidase EP 24.11, in contrast to the alpha-amino acid based parent peptide. This study has therefore clearly shown the potential of beta-amino acids in the design of stable enzyme inhibitors and their use in generating molecules with selectivity between closely related enzymes.

Amino Acid Substitution↗

PubMed2002

Soluble metalloendopeptidases and neuroendocrine signaling.

Peptidases play a vital and often highly specific role in the physiological and pathological generation and termination of peptide hormone signals. The thermolysin-like family of metalloendopeptidases involved in the extracellular processing of neuroendocrine and cardiovascular peptides are of particular significance, reflecting both their specificity for particular peptide substrates and their utility as therapeutic targets. Although the functions of the membrane-bound members of this family, such as angiotensin-converting enzyme and neutral endopeptidase, are well established, a role for the predominantly soluble family members in peptide metabolism is only just emerging. This review will focus on the biochemistry, cell biology, and physiology of the soluble metalloendopeptidases EC 3.4.24.15 (thimet oligopeptidase) and EC 3.4.24.16 (neurolysin), as well as presenting evidence that both peptidases play an important role in such diverse functions as reproduction, nociception, and cardiovascular homeostasis.

Amino Acid Sequence↗

PubMed2002

Identification of the 49-kDa autoantigen associated with lymphocytic hypophysitis as alpha-enolase.

Lymphocytic hypophysitis is part of the spectrum of organ-specific autoimmune diseases, and although its histopathology is well documented, its pathogenesis is unclear. Serum autoantibodies directed against a 49-kDa cytosolic protein are detected by immunoblotting in 70% of patients with biopsy-proven lymphocytic hypophysitis. Here we report the purification and identification of this first target autoantigen in lymphocytic hypophysitis. The autoantigen has a molecular mass of 49 kDa, a cytosolic localization, and a ubiquitous tissue distribution. The 49-kDa protein was purified from monkey brain and human placental cytosol. Limited amino acid sequencing after proteolytic digestion of the human placental protein showed identity with alpha-enolase. The identification was confirmed using sera from patients with pituitary autoimmunity, which strongly reacted with recombinant human alpha-enolase and yeast enolase, but not with rabbit muscle beta- enolase. This indicates that the immunoreactive epitopes are largely conserved from yeast to human, but are not present in beta-enolase. alpha-Enolase autoantibodies are not specific to pituitary autoimmune disease and have been reported in other autoimmune diseases. However, this study is the first to indicate a role for alpha-enolase as an autoantigen in lymphocytic hypophysitis.

Amino Acid Sequence↗

PubMed2002

Beta-amino acids: versatile peptidomimetics.

The use of peptidomimetics has emerged as a powerful means for overcoming the limitations inherent in the physical characteristics of peptides thus improving their therapeutic potential. A peptidomimetic approach that has emerged in recent years with significant potential, is the use of beta-amino acids. Beta-amino acids are similar to alpha-amino acids in that they contain an amino terminus and a carboxyl terminus. However, in beta-amino acids two carbon atoms separate these functional termini. beta-amino acids, with a specific side chain, can exist as the R or S isomers at either the alpha (C2) carbon or the beta (C3) carbon. This results in a total of 4 possible diastereoisomers for any given side chain. The flexibility to generate a vast range of stereo- and regioisomers, together with the possibility of disubstitution, significantly expands the structural diversity of beta-amino acids thereby providing enormous scope for molecular design. The incorporation of beta-amino acids has been successful in creating peptidomimetics that not only have potent biological activity, but are also resistant to proteolysis. This article reviews the rapidly expanding applications of beta-amino acids in the design of bioactive peptide analogues ranging from receptor agonists and antagonists, MHC-binding peptides, antimicrobial peptides and peptidase inhibitors. Given their structural diversity taken together with the ease of synthesis and incorporation into peptide sequences using standard solid-phase peptide synthesis techniques, beta-amino acids have the potential to form a new platform technology for peptidomimetic design and synthesis.

Amino Acids↗