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Volumn 16, Issue 3, 2015, Pages 415-423

Establishing the stability and reversibility of protein pyrophosphorylation with synthetic peptides

Author keywords

Phosphatases; Post translational modification; Pyrophosphorylation; Signaling mechanisms; Stability

Indexed keywords

ALKALINE PHOSPHATASE; CASEIN KINASE II; PHOSPHOPEPTIDE; PHOSPHOPROTEIN PHOSPHATASE 1; PYROPHOSPHATE; PYROPHOSPHOPEPTIDE; SERINE; SYNTHETIC PEPTIDE; THREONINE; UNCLASSIFIED DRUG; PHOSPHATASE; PYROPHOSPHORIC ACID DERIVATIVE;

EID: 84922569249     PISSN: 14394227     EISSN: 14397633     Source Type: Journal    
DOI: 10.1002/cbic.201402589     Document Type: Article
Times cited : (22)

References (72)
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    • note
    • When a more coordinating buffer (phosphate buffer) was used, no hydrolysis of any substrates was observed, presumably due to the buffer binding to the metal rather than to the peptide substrate. Therefore, the non-coordinating sulfonic-acid buffer, MOPS, was chosen, even though it exhibited modest background hydrolysis of the substrate. The background hydrolysis of the peptides in MOPS buffer alone was subtracted from the total hydrolysis of the peptides in MOPS with divalent metal cations present.
  • 44
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    • Y. Shi, Cell 2009, 139, 468-484.
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  • 48
    • 84922501389 scopus 로고    scopus 로고
    • note
    • We attribute the lack of hydrolysis of these phosphopeptides to their highly acidic nature, which might interfere with binding to these enzymes.
  • 49
    • 84922501388 scopus 로고    scopus 로고
    • note
    • Previous studies on in vitro pyrophosphorylated peptide and protein substrates by Snyder and co-workers[8b] accessed the effect of PP1 and PP2B, both of which are members of the PPP family and did not hydrolyze the pyrophosphate moiety. To our knowledge, the effect of PP2C, a PPM family member, on pyrophosphopeptide or protein hydrolysis had not previously been evaluated.
  • 50
    • 84922501387 scopus 로고    scopus 로고
    • note
    • The enzymatic activity of hDIPP1 was confirmed by treating P1P5-di(adenosine-5′)pentaphosphate(Ap5A), a known hDIPP1 substrate,[7d] with the enzyme, which resulted in 48% hydrolysis of Ap5A to adenosine monophosphate by HPLC after 30 min of incubation.
  • 51
    • 84922501386 scopus 로고    scopus 로고
    • note
    • Phosphopeptides 2, 6, 19 and pyrophosphopeptides 11, 15, 19 were treated with another small molecule pyrophosphatase, inorganic pyrophosphatase (iPPase), resulting in no hydrolysis of any of the peptide substrates, which is in agreement with previously published results.[8b]The activity of iPPase was confirmed by full hydrolysis of inorganic pyrophosphate to inorganic phosphate, as determined by 31P NMR under standard reaction conditions.
  • 54
    • 84922501385 scopus 로고    scopus 로고
    • note
    • No phosphopeptide intermediates were observed when the pyrophosphopeptides were treated with the AP enzymes under standard reaction conditions.
  • 55
    • 84922501384 scopus 로고    scopus 로고
    • note
    • As a small amount of the phosphopeptide intermediate was observed by HPLC (Figure S3), we believe that the hydrolysis in a S. cerevisiae lysate proceeds, at least in part, through a sequential dephosphorylation mechanism.
  • 61
    • 84922501383 scopus 로고    scopus 로고
    • note
    • These results were corroborated through treatment of 14P and 15P with the pho8Δ lysates, where a more prominent reduction in hydrolysis was observed (Figure S5C, S6C).
  • 62
    • 84922501382 scopus 로고    scopus 로고
    • note
    • We also tested the yeast enzyme most similar to Pho8 and Pho13[38] by treating pyrophosphopeptides 14P, 15P, and 19 and phosphopeptides 5P, 6P, and 10 with a pho13Δ lysate. In this case, the levels of hydrolysis did not decrease for either the pyrophospho- or phosphopeptide (Figure S7 A-C).
  • 66
    • 84922501381 scopus 로고    scopus 로고
    • note
    • Although this level of hydrolysis appears lower than for the yeast system, it is important to note that our standard assay conditions used a lower concentration of HeLa cell lysate (0.8 mg mL-1) than S. cerevisiae cell lysate (1.7 mg mL-1).
  • 71
    • 84922501380 scopus 로고    scopus 로고
    • note
    • We did not pursue genetic perturbations of APs in HeLa cells because HeLa cells express three related AP enzymes, complicating interpretation of knockdown studies.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.