Difference between revisions of "Team:SMMU-China/Description"

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The main function of the heart is to pump blood, providing power to promote blood circulation to meet the metabolic needs of tissue cells throughout the body.
 
The main function of the heart is to pump blood, providing power to promote blood circulation to meet the metabolic needs of tissue cells throughout the body.
 
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<h2 class="inner-h">2 CaRTIN</h2>
 
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<h3 class="inner-h">What is RyR2? What is it made up of?</h3>
 
 
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We first obtained and purified RyR2 from rat heart by using GST-fused FKBP12 as the published strategies described. To construct the camel VHH library, blood samples of 30 non-immunized, four-year-old male Bactrian camel were collected. B lymphocyte cDNA encoding VHHs was used to construct a phage display VHH library that consisted of approximately 3×10<sup>8</sup> individual colonies. VHH gene corresponded to the size of insert of over 98% colonies. For confirming the heterogeneity of the individual clones from the library, we sequenced fifty randomly selected clones, and each clone showed a distinct VHH sequence.
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Heart failure is when the heart's systolic or diastolic function declines, resulting in a failure of cardiac output to meet the body's metabolic needs. Heart failure is the severe and terminal stage of various heart diseases. According to the patients at all stages, patients including hypertension, coronary heart disease and diabetes are at risk of heart failure.
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In order to select nanobodies with specific ability to bind RyR2, bio-panning was performed with immobilized RyR2 protein. After the third round of panning, the result showed an obvious enrichment of phage particles that carried RyR2-specific VHH (Fig. 1A). Phage clones exhibited increased binding to RyR2 after the second round of panning. During four rounds of panning there was no phage clone that was found binding to BSA (Fig. 1B). VHH fragments of 300 individual colonies that were randomly chosen were expressed in an ELISA for screening colonies which bound to RyR2. Among these clones, 276 antibody fragments specifically bound to RyR2. One antibody fragment which did not bind to RyR2 was choose as a negative control, termed as VHH-AR117. To obtain antibodies that functionally inhibit of RyR2 phosphorylation, each of the antibody fragments was tested for its effect in an ELSA based RyR2 phosphorylation assay. 4 antibody fragments were potent inhibitors of RyR2 phosphorylation. The complementary determining regions (CDRs) were confirmed by sequence analysis and the result revealed that there was only one unique clone in this panel of antibody fragments, termed as VHH-AR185. To investigating the basis of dephosphorylation of RyR2 by VHH-AR185, the binding affinity of VHH-AR185 to RyR2 was measured by surface plasmon resonance. VHHs were purified for these experiments by expressing and secreting from the E. coli cytosol. As shown in Fig. 1D, the affinity (KD) of VHH-AR185 to RyR2 was estimated to be 1.93 nM. The result of affinity studies likely explained the inhibition of RyR2 phosphorylation due to the extremely slow dissociation rate of VHH-AR185 from RyR2.
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<strong>Figure 1. Isolation of RyR2-specific nanobody by phage display.</strong><br> (A) Phage-displayed nanobody fragments were selected against RyR2 by four rounds of panning. A gradual increase in phage titers was detected after each round of panning. (B) Polyclonal phage ELISA from the output phage of each round of panning. Control group used BSA as the irrelevant antigen. (C) Heat map generated from ELISA data of purified RyR2 channels which were phosphorylated in the presence of the PKA. (D) Kinetic analysis of AR185 binding to RyR2 was performed by SPR.
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<strong>Figure 1. </strong>
 
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The interaction of VHH-AR185 to RyR2 in the cytoplasm of eukaryotic cells was examined by co-immunoprecipitation experiments. VHH-AR185 and RyR2 were expressed in neonatal cardiomyocytes cells and the lysates of transfected cells were detected. As the result in fig. S1, anti-his antibody was able to efficiently co-precipitate RyR2 from the cells that expressed VHH-AR185-HIS, but could not co-precipitate RyR2 from cells expressing VHH-AR117-HIS. Conversely, anti-RyR2 antibody was able to co-precipitate VHH-AR185-HIS with RyR2, but not VHH-AR185-HIS. This result indicated that the VHH-AR185 could maintain its antigen binding ability in the cytoplasm and fold as a soluble protein.
 
 
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To identify the epitopes recognized by AR185, phage clones were isolated by panning the PhD.-7 phage display peptide library with AR185. Three rounds of selection were performed, and, at each round, the library was pre-cleared with a control AR177 nanobody. After the third round of panning, the binding of the isolated phage clones to AR185 was determined by ELISA. Sequence analysis of AR185-positive phage clones identified five and six distinct amino acid sequences, respectively (fig. S2). Alignment of these sequences revealed the consensus motifs DKLAC, which could be aligned with the (2725) DKLAN (2729) sequence located at P2 Domain of RyR2.
 
To identify the epitopes recognized by AR185, phage clones were isolated by panning the PhD.-7 phage display peptide library with AR185. Three rounds of selection were performed, and, at each round, the library was pre-cleared with a control AR177 nanobody. After the third round of panning, the binding of the isolated phage clones to AR185 was determined by ELISA. Sequence analysis of AR185-positive phage clones identified five and six distinct amino acid sequences, respectively (fig. S2). Alignment of these sequences revealed the consensus motifs DKLAC, which could be aligned with the (2725) DKLAN (2729) sequence located at P2 Domain of RyR2.
 
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Revision as of 16:11, 8 October 2018

1 Heart failure
2 CaRTIN
References

Description

1 Heart failure

What is heart failure?

The main function of the heart is to pump blood, providing power to promote blood circulation to meet the metabolic needs of tissue cells throughout the body.

Heart failure is when the heart's systolic or diastolic function declines, resulting in a failure of cardiac output to meet the body's metabolic needs. Heart failure is the severe and terminal stage of various heart diseases. According to the patients at all stages, patients including hypertension, coronary heart disease and diabetes are at risk of heart failure.

Figure 1.

2 CaRTIN

What is RyR2? What is it made up of?

To identify the epitopes recognized by AR185, phage clones were isolated by panning the PhD.-7 phage display peptide library with AR185. Three rounds of selection were performed, and, at each round, the library was pre-cleared with a control AR177 nanobody. After the third round of panning, the binding of the isolated phage clones to AR185 was determined by ELISA. Sequence analysis of AR185-positive phage clones identified five and six distinct amino acid sequences, respectively (fig. S2). Alignment of these sequences revealed the consensus motifs DKLAC, which could be aligned with the (2725) DKLAN (2729) sequence located at P2 Domain of RyR2.

References

  1. W. Chen et al., Outline of the report on cardiovascular diseases in China, 2014. 18, F2 (2016).
  2. B. Ziaeian, G. C. J. N. R. C. Fonarow, Epidemiology and aetiology of heart failure. 13, 368 (2016).
  3. C. W. Yancy et al., 2016 ACC/AHA/HFSA Focused Update on New Pharmacological Therapy for Heart Failure: An Update of the 2013 ACCF/AHA Guideline for the Management of Heart Failure. 68, 1476-1488 (2016).
  4. J. S. Hulot, K. Ishikawa, R. J. J. E. H. J. Hajjar, Gene therapy for the treatment of heart failure: promise postponed. 37, 1651-1658 (2016).
  5. M. Luo, M. E. J. C. R. Anderson, Mechanisms of Altered Ca^2^+ Handling in Heart Failure. 113, 690-708 (2013).
  6. F. V. J. J. o. B. C. Petegem, Ryanodine Receptors: Structure and Function. 287, 31624-31632 (2012).
  7. G. W. Cho, F. Altamirano, J. A. J. B.-M. B. o. D. Hill, Chronic heart failure: Ca2+, catabolism, and catastrophic cell death. 1862, 763-777 (2016).
  8. J. Roussel et al., Palmitoyl-carnitine increases RyR2 oxidation and sarcoplasmic reticulum Ca 2 + leak in cardiomyocytes: Role of adenine nucleotide translocase. 1852, 749-758 (2015).
  9. M. Munkvik et al., Training effects on skeletal muscle calcium handling in human chronic heart failure. 42, 847-855 (2010).
  10. S. O. Marx et al., PKA Phosphorylation Dissociates FKBP12.6 from the Calcium Release Channel (Ryanodine Receptor). 101, 365-376 (2000).
  11. X. H. Wehrens et al., Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression. Proc Natl Acad Sci U S A 103, 511-518 (2006).
  12. J. Shan et al., Role of chronic ryanodine receptor phosphorylation in heart failure and β-adrenergic receptor blockade in mice. 120, 4375-4387 (2010).
  13. H. Zhang et al., Hyperphosphorylation of the cardiac ryanodine receptor at serine 2808 is not involved in cardiac dysfunction after myocardial infarction. 110, 831 (2012).
  14. S. R. J. C. R. Houser, Role of RyR2 phosphorylation in heart failure and arrhythmias: protein kinase A-mediated hyperphosphorylation of the ryanodine receptor at serine 2808 does not alter cardiac contractility or cause heart failure and arrhythmias. 114, 1320-1327 (2014).
  15. S. Steeland, R. E. Vandenbroucke, C. J. D. D. T. Libert, Nanobodies as therapeutics: big opportunities for small antibodies. 21, 1076-1113 (2016).
  16. F. Peyvandi et al., Caplacizumab reduces the frequency of major thromboembolic events, exacerbations, and death in patients with acquired thrombotic thrombocytopenic purpura. 15, 1448-1452 (2017).
  17. T. Dörner et al., in European Congress of Rheumatology, 14–17 June. (2017), pp. 575.572-575.
  18. Safety and efficacy of multiple ascending doses of subcutaneous M1095, an anti-interleukin-17A/F bispecific nanobody, in patients with moderate-to-severe psoriasis %J Retour Au Numéro. (2017).
  19. L. D. Wang, D. X. Feng, S. M. Zhang, B. M. J. L. i. B. University, Research Progress of Nanobody. (2016).
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