Article

Sampling the N-terminal proteome of human blood

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 02/2010; 107(10):4561-6. DOI: 10.1073/pnas.0914495107
Source: PubMed

ABSTRACT The proteomes of blood plasma and serum represent a potential gold mine of biological and diagnostic information, but challenges such as dynamic range of protein concentration have hampered efforts to unlock this resource. Here we present a method to label and isolate N-terminal peptides from human plasma and serum. This process dramatically reduces the complexity of the sample by eliminating internal peptides. We identify 772 unique N-terminal peptides in 222 proteins, ranging over six orders of magnitude in abundance. This approach is highly suited for studying natural proteolysis in plasma and serum. We find internal cleavages in plasma proteins created by endo- and exopeptidases, providing information about the activities of proteolytic enzymes in blood, which may be correlated with disease states. We also find signatures of signal peptide cleavage, coagulation and complement activation, and other known proteolytic processes, in addition to a large number of cleavages that have not been reported previously, including over 200 cleavages of blood proteins by aminopeptidases. Finally, we can identify substrates from specific proteases by exogenous addition of the protease combined with N-terminal isolation and quantitative mass spectrometry. In this way we identified proteins cleaved in human plasma by membrane-type serine protease 1, an enzyme linked to cancer progression. These studies demonstrate the utility of direct N-terminal labeling by subtiligase to identify and characterize endogenous and exogenous proteolysis in human plasma and serum.

0 Bookmarks
 · 
68 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Staphylococcus aureus produces peptide toxins that it uses to respond to environmental cues. We previously characterized PepA1, a peptide toxin from S. aureus, that induces lytic cell death of both bacterial and host cells. That led us to suggest that PepA1 has an antibacterial activity. Here, we demonstrate that exogenously provided PepA1 has activity against both Gram-positive and Gram-negative bacteria. We also see that PepA1 is significantly hemolytic, thus limiting its use as an antibacterial agent. To overcome these limitations, we converted PepA1 into nonhemolytic derivatives. Our most promising derivative is a cyclic heptapseudopeptide with inconsequential toxicity to human cells, enhanced stability in human sera, and sharp antibacterial activity. Mechanistically, linear and helical PepA1 derivatives form pores at the bacterial and erythrocyte surfaces, while the cyclic peptide induces bacterial envelope reorganization, with insignificant action on the erythrocytes. Our work demonstrates that bacterial toxins might be an attractive starting point for antibacterial drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, however, the underlying mechanisms remain unknown. We used a dedicated N-terminomics technique, iTRAQ-TAILS (Terminal Amine Isotopic Labeling of Substrates), to characterize the human platelet proteome, N-terminome, and post-translational modifications throughout platelet storage over 9 days under blood banking conditions. From the identified 2,938 proteins and 7,503 unique peptides we characterized N-terminal methionine excision, co- and post-translational Nα-acetylation, protein maturation and proteolytic processing of proteins in human platelets. We also identified for the first time in platelets 10 proteins previously classified by HUPO as "missing" in the human proteome. Most of N-termini (77%) were internal neo-N-termini: 105 were novel potential alternative translation start sites; and 2,180 represented stable proteolytic products, thus highlighting a prominent yet previously uncharacterized role of proteolytic processing during platelet storage. Protease inhibitor studies revealed metalloproteinases as being primarily responsible for proteolytic processing (as opposed to degradation) during storage. System-wide identification of metallo- and other proteinase substrates and their respective cleavage sites suggests novel mechanisms of the effect of proteases on protein activity and platelet function during storage. All datasets and metadata are available through ProteomeXchange with the dataset identifier PXD000906.
    Blood 10/2014; 124(26). DOI:10.1182/blood-2014-04-569640 · 9.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function-first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100-400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence.
    Scientific Reports 09/2014; 4:6008. DOI:10.1038/srep06008 · 5.08 Impact Factor

Preview

Download
0 Downloads
Available from