Expression of Chaperone–Synuclein Fusion Proteins and Their Regulatory Effects on Amyloid Fibril Formation

Kim, Soohyun; Shin, Eunsook; Im, Hana; Lee, Kyung‐Hee

doi:10.1002/bkcs.10546

Cited by 3 publications

(4 citation statements)

References 20 publications

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“…We tested if the fusion proteins (FKBP‐L23‐hIAPP and FKBPIF‐L23‐hIAPP) possess either the PPIase activity or chaperone activity as demonstrated in the system of Syn‐fused proteins in our previous study . To examine the PPIase activity of FKBP or FKBPIF domain as part of a fusion protein, we carried out a chymotrypsin‐coupled PPIase assay .…”

Section: Resultsmentioning

confidence: 99%

“…Proteins FKBP and FKBPIF were expressed and purified using FKBP/pET28a and FKBPIF/pET28a plasmids as previously described . Fusion protein‐encoding plasmid FKBP‐L23‐hIAPP/pET28a was constructed using cassette ligation starting from FKBP‐L23‐Syn/pET28a as previously described . Briefly, the DNA fragment encoding hIAPP with BamH1/XhoI sites at the ends was subcloned into the BamH1/Xho1‐digested vector part of FKBP‐L23‐Syn/pET28a.…”

Section: Methodsmentioning

confidence: 99%

“…We constructed the fusion plasmids carrying FKBP (or FKBPIF) as well as hIAPP in a bacterial expression vector, pET28a. Starting from FKBP‐L23‐Syn/pET28a plasmid, which has been constructed in our laboratory, FKBP‐L23‐hIAPP/pET28a was assembled by cassette ligation . The BamH1/Xho1‐double digested‐hIAPP gene amplified by PCR was connected by T4 DNA ligase to the cognate fragment of FKBP‐L23‐Syn/pET28a.…”

Section: Methodsmentioning

confidence: 99%

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Regulation of Amyloid Fibril Formation from Human Islet Amyloid Polypeptide by a Ligand Binding to the Fusion of FK506‐binding Protein and the Insertion‐in‐Flap Domain

Yoon

Kim

et al. 2017

Bulletin Korean Chem Soc

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Human islet amyloid polypeptide (hIAPP) is converted to toxic aggregates in type‐2 diabetes (T2D). With the aim of developing a candidate agent regulating hIAPP amyloid fibril formation, we constructed a series of recombinant fusion proteins using a bacterial expression system. FKBPIF‐L23‐hIAPP and F36VIF‐L23‐hIAPP accelerated amyloid fibril formation. Assays of cis/trans‐peptidyl‐prolyl isomerase activity as well as chaperone activity of FKBPIF‐L23‐hIAPP fusion proteins suggested that amyloid fibrils were produced because the chaperone activity of FKBPIF in the form of fusion protein was diminished. Moreover, addition of FK506, a binding ligand, slowed down the process of amyloid fibril formation in FKBPIF‐L23‐hIAPP fusion protein. Deletion mutant analysis of the insertion‐in‐flap (IF) domain revealed that the C‐terminal half is essential for acceleration of amyloid fibril formation. Based on these observations, we suggested a potential anti‐T2D therapeutic strategy by means of a small ligand and fragments of the IF domain inserted in a fusion protein.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Regulation of Amyloid Fibril Formation from Human Islet Amyloid Polypeptide by a Ligand Binding to the Fusion of FK506‐binding Protein and the Insertion‐in‐Flap Domain

Yoon

Kim

et al. 2017

Bulletin Korean Chem Soc

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“…Once bound, the presence of IF in the FKBPIF makes αSyn form amyloid fibril at a faster rate than FKBP itself. These observations implied that it might extend the helical structure of αSyn, resulting in a more aggregation‐prone structure . Notably, the modulation of amyloid fibril formation of αSyn was a ligand‐specific reversible process, which was regulated by a small organic molecule, like FK506 or Shield‐1 …”

Section: Introductionmentioning

confidence: 99%

Amyloid Fibril Formation of α‐Synuclein Is Modulated via the Estrogen Receptor Ligand Binding Domain of Estrogen Receptor α Bound with Tamoxifen‐based Small Molecules

Lee

Yoon

Lee

et al. 2020

Bulletin Korean Chem Soc

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Alpha‐synuclein (αSyn) forms amyloid fibrils in the brain of patients suffering from Parkinson's disease. Aiming at the development of a new therapy via regulating amyloid fibril formation of αSyn, we studied estrogen receptor ligand‐binding domain (ERLBD) of estrogen receptor α (ΕRα). The ERLBD encoding gene was cloned, expressed by IPTG induction, and successfully purified by Ni2+‐affinity chromatography followed by anion‐exchange chromatography. In the thioflavin T (ThT) fluorescence assay, we examined the kinetics of amyloid fibril formation of αSyn in the presence of ERLBD protein. Dose‐dependent inhibition of amyloid fibril formation of αSyn by ERLBD protein was observed, indicating that ERLBD protein binds to αSyn, thereby blocking amyloid fibril formation. The kinetics of amyloid fibril formation of αSyn were compared in the absence and presence of a small molecule, such as 17β‐estradiol (E2, agonist) or tamoxifen (TMX, antagonist) of ΕRα. Regardless of the presence of E2, we found that ERLBD protein inhibits αSyn from forming amyloid fibrils. On the other hand, TMX reversed the inhibition effect of ERLBD protein on amyloid fibril formation of αSyn. Interestingly, the photo‐reactive hybrid ligand 2‐phenylquinoline‐4‐hydroxytamoxifen (PQ‐OHT) was expected to exhibit the anti‐inhibitory effect of ERLBD on amyloid fibril formation of αSyn. Upon illuminating with a UV lamp, PQ‐OHT was found to degrade ERLBD protein as well. These observations suggest the examination of the possibility that a complex of ERLBD‐αSyn amyloid fibril can be eliminated via the ligand PQ‐OHT upon exposure to light for therapeutic purposes.

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Modified and environmentally friendly oxidation of phosphorane ylides to vicinal tricarbonyls using unsupported moist Oxone in dichloromethane

Lee

2020

Synthetic Communications

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Expression of Chaperone–Synuclein Fusion Proteins and Their Regulatory Effects on Amyloid Fibril Formation

Cited by 3 publications

References 20 publications

Regulation of Amyloid Fibril Formation from Human Islet Amyloid Polypeptide by a Ligand Binding to the Fusion of FK506‐binding Protein and the Insertion‐in‐Flap Domain

Regulation of Amyloid Fibril Formation from Human Islet Amyloid Polypeptide by a Ligand Binding to the Fusion of FK506‐binding Protein and the Insertion‐in‐Flap Domain

Amyloid Fibril Formation of α‐Synuclein Is Modulated via the Estrogen Receptor Ligand Binding Domain of Estrogen Receptor α Bound with Tamoxifen‐based Small Molecules

Modified and environmentally friendly oxidation of phosphorane ylides to vicinal tricarbonyls using unsupported moist Oxone in dichloromethane

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