Removal of N-terminal methionine from haemoglobin nascent peptides by a membrane-bound rat liver methionine aminopeptidase.
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ABSTRACT:
SUBMITTER: Termignoni C
PROVIDER: S-EPMC1146589 | biostudies-other | 1986 Mar
REPOSITORIES: biostudies-other
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Project description:The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.
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Project description:Quantifying the number of charges on peptides bound to interfaces requires reliable estimates of (i) surface coverage and (ii) surface charge, both of which are notoriously difficult parameters to obtain, especially at solid/water interfaces. Here, we report the thermodynamics and electrostatics governing the interactions of l-lysine and l-arginine octamers (Lys8 and Arg8) with supported lipid bilayers prepared from a 9?:?1 mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DMPG) from second harmonic generation (SHG) spectroscopy, quartz crystal microbalance with dissipation monitoring (QCM-D) and nanoplasmonic sensing (NPS) mass measurements, and atomistic simulations. The combined SHG/QCM-D/NPS approach provides interfacial charge density estimates from mean field theory for the attached peptides that are smaller by a factor of approximately two (0.12 ± 0.03 C m-2 for Lys8 and 0.10 ± 0.02 C m-2 for Arg8) relative to poly-l-lysine and poly-l-arginine. These results, along with atomistic simulations, indicate that the surface charge density of the supported lipid bilayer is neutralized by the attached cationic peptides. Moreover, the number of charges associated with each attached peptide is commensurate with those found in solution; that is, Lys8 and Arg8 are fully ionized when attached to the bilayer. Computer simulations indicate Lys8 is more likely than Arg8 to "stand-up" on the surface, interacting with lipid headgroups through one or two sidechains while Arg8 is more likely to assume a "buried" conformation, interacting with the bilayer through up to six sidechains. Analysis of electrostatic potential and charge distribution from atomistic simulations suggests that the Gouy-Chapman model, which is widely used for mapping surface potential to surface charge, is semi-quantitatively valid; despite considerable orientational preference of interfacial water, the apparent dielectric constant for the interfacial solvent is about 30, due to the thermal fluctuation of the lipid-water interface.
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Project description:Bacterial infection is one of the leading causes of death in young, elderly, and immune-compromised patients. The rapid spread of multi-drug-resistant (MDR) bacteria is a global health emergency and there is a lack of new drugs to control MDR pathogens. We describe a heretofore-unexplored discovery pathway for novel antibiotics that is based on self-targeting, structure-disrupting peptides. We show that a helical peptide, KFF- EcH3, derived from the Escherichia coli methionine aminopeptidase can disrupt secondary and tertiary structure of this essential enzyme, thereby killing the bacterium (including MDR strains). Significantly, no detectable resistance developed against this peptide. Based on a computational analysis, our study predicted that peptide KFF- EcH3 has the strongest interaction with the structural core of the methionine aminopeptidase. We further used our approach to identify peptide KFF- NgH1 to target the same enzyme from Neisseria gonorrhoeae. This peptide inhibited bacterial growth and was able to treat a gonococcal infection in a human cervical epithelial cell model. These findings present an exciting new paradigm in antibiotic discovery using self-derived peptides that can be developed to target the structures of any essential bacterial proteins.-Zhan, J., Jia, H., Semchenko, E. A., Bian, Y., Zhou, A. M., Li, Z., Yang, Y., Wang, J., Sarkar, S., Totsika, M., Blanchard, H., Jen, F. E.-C., Ye, Q., Haselhorst, T., Jennings, M. P., Seib, K. L., Zhou, Y. Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.
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Project description:Excision of the N-terminal initiator methionine (iMet) residue from nascent peptide chains is an essential and omnipresent protein modification carried out by methionine aminopeptidases (MetAPs) that accounts for a major source of N-terminal proteoform diversity. While MetAP2 is known to be implicated in processes such as angiogenesis and proliferation in mammals, the physiological role of MetAP1 is much less clear. In this report we studied the omics-wide effects of human MetAP1 deletion and general MetAP inhibition. The levels of iMet retention are inversely correlated with cellular proliferation rates. Further, despite the increased MetAP2 expression observed upon MetAP1 deletion and as inferred from the iMet retention profiles observed, MetAP2 was unable to restore processing of MS-, MP- and MA- N-termini, indicating the higher activity of MetAP1 over these N-termini. Proteome and transcriptome expression profiling point to differential expression of proteins implicated in lipid metabolism, cytoskeleton organization, cell proliferation and protein synthesis upon perturbation of MetAP activity.
2018-01-12 | GSE103405 | GEO
Project description:The mammalian nuclear lamina proteins—prelamin A- and B-type lamins—are post-translationally modified by farnesylation, endoproteolysis, and carboxymethylation at a carboxy-terminal CAAX (C, cysteine; a, aliphatic amino acid; X, any amino acid) motif. However, prelamin A processing into mature lamin A is a unique process because it results in the production of farnesylated and carboxymethylated peptides. In cells from patients with Hutchinson⁻Gilford progeria syndrome, the mutant prelamin A protein, progerin, cannot release its prenylated carboxyl-terminal moiety and therefore remains permanently associated with the nuclear envelope (NE), causing severe nuclear alterations and a dysmorphic morphology. To obtain a better understanding of the abnormal interaction and retention of progerin in the NE, we analyzed the spatiotemporal distribution of the EGFP fusion proteins with or without a nuclear localization signal (NLS) and a functional CAAX motif in HeLa cells transfected with a series of plasmids that encode the carboxy-terminal ends of progerin and prelamin A. The farnesylated carboxy-terminal fusion peptides bind to the NE and induce the formation of abnormally shaped nuclei. In contrast, the unfarnesylated counterparts exhibit a diffuse localization in the nucleoplasm, without obvious NE deformation. High levels of farnesylated prelamin A and progerin carboxy-terminal peptides induce nucleophagic degradation of the toxic protein, including several nuclear components and chromatin. However, SUN1, a constituent of the linker of nucleoskeleton and cytoskeleton (LINC) complex, is excluded from these autophagic NE protrusions. Thus, nucleophagy requires NE flexibility, as indicated by SUN1 delocalization from the elongated NE⁻autophagosome complex.
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Project description:Methionine aminopeptidases (MetAPs) remove the initiator methionine during protein biosynthesis. They exist in two isoforms, MetAP1 and MetAP2. The anti-angiogenic compound fumagillin binds tightly to the Type 2 MetAPs but only weakly to Type 1. High-affinity complexes of fumagillin and its relative ovalicin with Type 2 human MetAP have been reported. Here we describe the crystallographic structure of the low-affinity complex between ovalicin and Type 1 human MetAP at 1.1 A resolution. This provides the first opportunity to compare the structures of ovalicin or fumagillin bound to a Type 1 and a Type 2 MetAP. For both Type 1 and Type 2 human MetAPs the inhibitor makes a covalent adduct with a corresponding histidine. At the same time there are significant differences in the alignment of the inhibitors within the respective active sites. It has been argued that the lower affinity of ovalicin and fumagillin for the Type 1 MetAPs is due to the smaller size of their active sites relative to the Type 2 enzymes. Comparison with the uncomplexed structure of human Type 1 MetAP indicates that there is some truth to this. Several active site residues have to move "outward" by 0.5 Angstroms or so to accommodate the inhibitor. Other residues move "inward." There are, however, other factors that come into play. In particular, the side chain of His310 rotates by 134 degrees into a different position where (together with Glu128 and Tyr195) it coordinates a metal ion not seen at this site in the native enzyme.
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Project description:Excision of the N-terminal initiator methionine (iMet) residue from nascent peptide chains is an essential and omnipresent protein modification carried out by methionine aminopeptidases (MetAPs) that accounts for a major source of N-terminal proteoform diversity. Although MetAP2 is known to be implicated in processes such as angiogenesis and proliferation in mammals, the physiological role of MetAP1 is much less clear. In this report we studied the omics-wide effects of human MetAP1 deletion and general MetAP inhibition. The levels of iMet retention are inversely correlated with cellular proliferation rates. Further, despite the increased MetAP2 expression on MetAP1 deletion, MetAP2 was unable to restore processing of Met-Ser-, Met-Pro-, and Met-Ala- starting N termini as inferred from the iMet retention profiles observed, indicating a higher activity of MetAP1 over these N termini. Proteome and transcriptome expression profiling point to differential expression of proteins implicated in lipid metabolism, cytoskeleton organization, cell proliferation and protein synthesis upon perturbation of MetAP activity.
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Project description:Methionine aminopeptidases (MetAPs) are metalloenzymes that cleave the N-terminal methionine from newly synthesized peptides and proteins. These MetAP enzymes are present in bacteria, and knockout experiments have shown that MetAP activity is essential for cell life, suggesting that MetAPs are good antibacterial drug targets. MetAP enzymes are also present in the human host and selectivity is essential. There have been significant structural biology efforts and over 65 protein crystal structures of bacterial MetAPs are deposited into the PDB. This review highlights the available crystallographic data for bacterial MetAPs. Structural comparison of bacterial MetAPs with human MetAPs highlights differences that can lead to selectivity. In addition, this review includes the chemical diversity of molecules that bind and inhibit the bacterial MetAP enzymes. Analysis of the structural biology and chemical space of known bacterial MetAP inhibitors leads to a greater understanding of this antibacterial target and the likely development of potential antibacterial agents.
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Project description:The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.
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Project description:Cellular protein synthesis is initiated with methionine in eukaryotes with few exceptions. Methionine aminopeptidases (MetAPs) which catalyze the process of N-terminal methionine excision are essential for all organisms. In mammals, type 2 MetAP (MetAP2) is known to be important for angiogenesis, while type 1 MetAP (MetAP1) has been shown to play a pivotal role in cell proliferation. Our previous high-throughput screening of a commercial compound library uncovered a novel class of inhibitors for both human MetAP1 (HsMetAP1) and human MetAP2 (HsMetAP2). This class of inhibitors contains a pyridinylpyrimidine core. To understand the structure-activity relationship (SAR) and to search for analogues of 2 with greater potency and higher HsMetAP1-selectivity, a total of 58 analogues were acquired through either commercial source or by in-house synthesis and their inhibitory activities against HsMetAP1 and HsMetAP2 were determined. Through this systematic medicinal chemistry analysis, we have identified (1) 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine as the minimum element for the inhibition of HsMetAP1; (2) 5'-chloro as the favored substituent on the pyridine ring for the enhanced potency against HsMetAP1; and (3) long C4 side chains as the essentials for higher HsMetAP1-selectivity. At the end of our SAR campaign, 25b, 25c, 26d and 30a-30c are among the most selective and potent inhibitors of purified HsMetAP1 reported to date. In addition, we also performed crystallographic analysis of one representative inhibitor (26d) in complex with N-terminally truncated HsMetAP1.
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