Protein splicing and autoproteolysis mechanisms

Perler, Francine B.; Xu, Ming‐Qun; Paulus, Henry

doi:10.1016/s1367-5931(97)80065-8

Cited by 145 publications

(115 citation statements)

References 29 publications

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“…For example, it has been demonstrated that intein-based mechanisms, which are normally used for the autocatalytic splicing of two linear protein domains (29), can be adapted for the production of artificial circular proteins, both in vitro and in vivo (30)(31)(32). Inspection of the sequences of the precursor proteins described here does not reveal any homologies with known intein sequences, all of which are substantially larger.…”

Section: Discussionmentioning

confidence: 82%

Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Jennings

West

Waine

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

479

679

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Several members of the Rubiaceae and Violaceae families produce a series of cyclotides or macrocyclic peptides of 29 -31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata B1 as well as three other clones for related cyclotides from the African plant Oldenlandia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46 -68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cyclotides have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

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Section: Discussionmentioning

confidence: 82%

Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Jennings

West

Waine

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

479

679

View full text Add to dashboard Cite

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“…Another possibility is that some of the intein splice product is abnormal and becomes a rapidly degraded DRiP. This is also unlikely because the intein splicing mechanism is quite precise (27) and we show that the resulting splice product is not unstable. Nevertheless, to further address these possibilities we evaluated whether DRiPs could be detected biochemically either before or after splicing of our intein constructs.…”

Section: Discussionmentioning

confidence: 83%

“…4C). Again the presentation of the HY epitope was similar (27). (C-F) Schematic of lentiviral construct inserts (note that the product of the prespliced construct is the same as generated by intein splicing).…”

Section: Resultsmentioning

confidence: 94%

Using intein catalysis to probe the origin of major histocompatibility complex class I-presented peptides

Farfán-Arribas

Stern

Rock

2012

Proc. Natl. Acad. Sci. U.S.A.

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All vertebrate nucleated cells generate peptides from their expressed gene products and then display them at the cell surface bound to MHC class I molecules. This allows CD8 + T cells to detect and eliminate abnormal cells that are synthesizing foreign proteins, e.g., from viruses or mutations. To permit the immune system to more uniformly monitor a cell's proteins, regardless of their half-life or location, it has been thought that the products of rapid degradation of the mistakes of protein synthesis (defective ribosomal products, DRiPs) preferentially contribute to the class I-presented peptides. However, using intein catalysis to generate peptide sequences exclusively by posttranslational splicing of mature proteins, we show here that presented peptides can be generated from fully folded and functional proteins. Remarkably, the presentation of peptides from two model mature proteins is just as efficient as from newly synthesized proteins subject to errors in translation or folding. These results indicate that for the constructs we have analyzed, DRiPs are not a more efficient source of class I peptides for antigen presentation than the turnover of mature functional proteins. Accordingly, our data suggest that one of the major ways the immune system evaluates the health of cells is by monitoring the breakdown products of the proteome.

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“…The proposed mechanism for the S. cerevisiae Vma intein is shown in Figure 3 (see refs. 24,25). Splicing is initiated by an N-S acyl shift that converts a peptide bond into a thioester at the N-extein junction site.…”

Section: Problem Under Investigationmentioning

confidence: 99%

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

2007

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Expressed protein ligation (EPL) allows semisynthesis of a target protein with site-specific incorporation of probes or unnatural amino acids at its N or C termini. Here, we describe the protocol that our lab has developed for incorporating fluorotyrosines (F n Ys) at residue 356 of the small subunit of Escherichia coli ribonucleotide reductase using EPL. In this procedure, the majority of the protein (residues 1-353 out of 375) is fused to an intein domain and prepared by recombinant expression, yielding the protein in a thioester-activated, truncated form. The remainder of the protein, a 22-mer peptide, is prepared by solid-phase peptide synthesis and contains the F n Y at the desired position. Ligation of the 22-mer peptide to the thioester-activated R2 and subsequent purification yield full-length R2 with the F n Y at residue 356. The procedure to generate 100 mg quantities of Y 356 F n Y-R2 takes 3-4 months. INTRODUCTION Problem under investigationThe Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of all four nucleotides to their corresponding 2¢-deoxynucleotides and consists of two homodimeric subunits: R1 and R2 (refs. 1-3). R1 is the homodimeric subunit where nucleotide reduction occurs. It contains binding sites for nucleoside diphosphate substrates as well as for deoxynucleoside triphosphate and ATP allosteric effectors, which govern substrate specificity and turnover rates. R2 is the homodimeric subunit, which houses the diiron-tyrosyl radical cofactor (Y 122 ) essential for catalysis. Each turnover requires radical migration from the Y 122 site on R2 to the active site of R1, over a distance of 35 Å . The mechanism of this long-range radical propagation event is an active area of research 4,5 .Structures of active RNR, a 1:1 complex of R1 and R2, are not available. However, Uhlin and Eklund 6 have generated a docking model of this complex from the individual structures of R1 and R2 (refs. 7,8

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Protein splicing and autoproteolysis mechanisms

Cited by 145 publications

References 29 publications

Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Using intein catalysis to probe the origin of major histocompatibility complex class I-presented peptides

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

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