Publications by authors named "Diana C Rodriguez Camargo"

11 Publications

  • Page 1 of 1

Expression, purification and characterisation of large quantities of recombinant human IAPP for mechanistic studies.

Biophys Chem 2021 Feb 28;269:106511. Epub 2020 Nov 28.

Department of Biophysical, Chemistry, Chemical Centre, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden; Wren Therapeutics Limited, UK. Electronic address:

Malfunction and amyloid formation of the Islet Amyloid Polypeptide (IAPP) are factors contributing to Type 2 diabetes. Unravelling the mechanism of IAPP aggregate formation may forward our understanding of this process and its effect on pancreatic β-islet cell. Such mechanistic studies require access to sequence homogeneous and highly pure IAPP. Here we present a new facile protocol for the production of pure recombinant human IAPP at relatively high yield. The protocol uses a His-tagged version of the N mutant EDDIE, which drives expression to inclusion bodies, from which the peptide is purified using sonication, refolding and auto-cleavage, removal of EDDIE using Ni-NTA chromatography and reverse-phase HPLC. The purified material is used at multiple concentrations in aggregation kinetics measurements monitored by thioflavin-T fluorescence. Global analysis of the data implies a double nucleation aggregation mechanism including both primary and secondary nucleation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpc.2020.106511DOI Listing
February 2021

Small molecule induced toxic human-IAPP species characterized by NMR.

Chem Commun (Camb) 2020 Nov 2;56(86):13129-13132. Epub 2020 Oct 2.

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective, which is reflected in a strong interference with hIAPP aggregation while showing weaker interactions with human-calcitonin and amyloid-β in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0cc04803hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641245PMC
November 2020

Microsecond motions probed by near-rotary-resonance RN MAS NMR experiments: the model case of protein overall-rocking in crystals.

J Biomol NMR 2018 05 30;71(1):53-67. Epub 2018 May 30.

Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.

Solid-state near-rotary-resonance measurements of the spin-lattice relaxation rate in the rotating frame (R) is a powerful NMR technique for studying molecular dynamics in the microsecond time scale. The small difference between the spin-lock (SL) and magic-angle-spinning (MAS) frequencies allows sampling very slow motions, at the same time it brings up some methodological challenges. In this work, several issues affecting correct measurements and analysis of N R data are considered in detail. Among them are signal amplitude as a function of the difference between SL and MAS frequencies, "dead time" in the initial part of the relaxation decay caused by transient spin-dynamic oscillations, measurements under HORROR condition and proper treatment of the multi-exponential relaxation decays. The multiple N R measurements at different SL fields and temperatures have been conducted in 1D mode (i.e. without site-specific resolution) for a set of four different microcrystalline protein samples (GB1, SH3, MPD-ubiquitin and cubic-PEG-ubiquitin) to study the overall protein rocking in a crystal. While the amplitude of this motion varies very significantly, its correlation time for all four sample is practically the same, 30-50 μs. The amplitude of the rocking motion correlates with the packing density of a protein crystal. It has been suggested that the rocking motion is not diffusive but likely a jump-like dynamic process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10858-018-0191-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986846PMC
May 2018

hIAPP forms toxic oligomers in plasma.

Chem Commun (Camb) 2018 May;54(43):5426-5429

Institute for Advanced Study, Technische Universität München, 85748 Garching, Germany.

In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in β-sheet rich oligomers with increased β-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c8cc03097aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970100PMC
May 2018

Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice.

Sci Rep 2018 01 18;8(1):1116. Epub 2018 Jan 18.

Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany.

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-18807-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773570PMC
January 2018

Stabilization and structural analysis of a membrane-associated hIAPP aggregation intermediate.

Elife 2017 11 17;6. Epub 2017 Nov 17.

Institute for Advanced Study, Technische Universität München, Garching, Germany.

Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique β-strand structure distinct from the conventional amyloid β-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.31226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706959PMC
November 2017

Limits of Resolution and Sensitivity of Proton Detected MAS Solid-State NMR Experiments at 111 kHz in Deuterated and Protonated Proteins.

Sci Rep 2017 08 7;7(1):7444. Epub 2017 Aug 7.

Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.

MAS solid-state NMR is capable of determining structures of protonated solid proteins using proton-detected experiments. These experiments are performed at MAS rotation frequency of around 110 kHz, employing 0.5 mg of material. Here, we compare H, C correlation spectra obtained from protonated and deuterated microcrystalline proteins at MAS rotation frequency of 111 kHz, and show that the spectral quality obtained from deuterated samples is superior to those acquired using protonated samples in terms of resolution and sensitivity. In comparison to protonated samples, spectra obtained from deuterated samples yield a gain in resolution on the order of 3 and 2 in the proton and carbon dimensions, respectively. Additionally, the spectrum from the deuterated sample yields approximately 2-3 times more sensitivity compared to the spectrum of a protonated sample. This gain could be further increased by a factor of 2 by making use of stereospecific precursors for biosynthesis. Although the overall resolution and sensitivity of H, C correlation spectra obtained using protonated solid samples with rotation frequencies on the order of 110 kHz is high, the spectral quality is still poor when compared to the deuterated samples. We believe that experiments involving large protein complexes in which sensitivity is limiting will benefit from the application of deuteration schemes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-07253-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547042PMC
August 2017

The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes.

Sci Rep 2017 03 13;7:44041. Epub 2017 Mar 13.

Helmholtz Zentrum München, Ingolstädter Landstr. 1, Neuherberg 85764, Germany.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep44041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347123PMC
March 2017

Restoring Resolution in Biological Solid-State NMR under Conditions of Off-Magic-Angle Spinning.

J Phys Chem Lett 2015 Dec 7;6(24):5040-4. Epub 2015 Dec 7.

Helmholtz-Zentrum München (HMGU) , Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany.

Spin-state-selective excitation (S3E) experiments allow the selection of individual transitions in a coupled two spin system. We show that in the solid state, the dipole-dipole interaction (DD) between (15)N and (1)H in a (1)H-(15)N bond and the chemical shift anisotropy (CSA) of (15)N in an amide moiety mutually cancel each other for a particular multiplet component at high field, when the sample is spun off the magic angle (Arctan [√2] = 54.74°). The accuracy of the adjustment of the spinning angle is crucial in conventional experiments. We demonstrate that for S3E experiments, the requirement to spin the sample exactly at the magic angle is not mandatory. Applications of solid state NMR in narrow bore magnets will be facilitated where the adjustment of the magic angle is often difficult. The method opens new perspectives for the development of schemes to determine distances and to quantify dynamics in the solid state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jpclett.5b02467DOI Listing
December 2015

Cloning, expression and purification of the human Islet Amyloid Polypeptide (hIAPP) from Escherichia coli.

Protein Expr Purif 2015 Feb 6;106:49-56. Epub 2014 Nov 6.

Helmholtz Zentrum München (Deutsches Forschungszentrum für Gesundheit und Umwelt), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universität München (TUM), Lichtenbergstr. 4, 85747 Garching, Germany. Electronic address:

Type II diabetes is characterized by deposition of the hormone human Islet Amyloid Polypeptide (hIAPP). Formation of hIAPP amyloid fibrils and aggregates is considered to be responsible for pancreatic β-cell losses. Therefore, insight into the structure of hIAPP in the solid-state and in solution is of fundamental importance in order to better understand the action of small molecules, which can potentially dissolve protein aggregates and modulate cell toxicity. So far, no procedure has been described that allows to obtain the native human IAPP peptide at high yields. We present here a cloning, expression and purification protocol that permits the production of 2.5 and 3mg of native peptide per liter of minimal and LB medium, respectively. In the construct, hIAPP is fused to a chitin binding domain (CBD). The CBD is subsequently cleaved off making use of intein splicing reaction which yield amidation of the C-terminus. The N-terminus contains a solubilization domain which is cleaved by V8 protease, avoiding additional residues at the N-terminus. The correct formation of the disulfide bond is achieved by oxidation with H2O2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pep.2014.10.012DOI Listing
February 2015

The FKBP-rapamycin binding domain of human TOR undergoes strong conformational changes in the presence of membrane mimetics with and without the regulator phosphatidic acid.

Biochemistry 2012 Jun 5;51(24):4909-21. Epub 2012 Jun 5.

Biomolecular NMR Spectroscopy, Department of Chemistry, Technische Universität München, Munich, Germany.

The Ser/Thr kinase target of rapamycin (TOR) is a central controller of cellular growth and metabolism. Misregulation of TOR signaling is involved in metabolic and neurological disorders and tumor formation. TOR can be inhibited by association of a complex of rapamycin and FKBP12 to the FKBP12-rapamycin binding (FRB) domain. This domain was further proposed to interact with phosphatidic acid (PA), a lipid second messenger present in cellular membranes. Because mammalian TOR has been localized at various cellular membranes and in the nucleus, the output of TOR signaling may depend on its localization, which is expected to be influenced by the interaction with complex partners and regulators in response to cellular signals. Here, we present a detailed characterization of the interaction of the FRB domain with PA and how it is influenced by the surrounding membrane environment. On the basis of nuclear magnetic resonance- and circular dichroism-monitored binding studies using different neutral and negatively charged lipids as well as different membrane mimetics (micelles, bicelles, and liposomes), the FRB domain may function as a conditional peripheral membrane protein. However, the data for the isolated domain just indicate an increased affinity for negatively charged lipids and membrane patches but no specific preference for PA or PA-enriched regions. The membrane-mimetic environment induces strong conformational changes that largely maintain the α-helical secondary structure content but presumably disperse the helices in the lipidic environment. Consistent with overlapping binding surfaces for different lipids and the FKBP12-rapamycin complex, binding of the inhibitor complex protects the FRB domain from interactions with membrane mimetics at lower lipid concentrations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi3002133DOI Listing
June 2012