Solution Structure Analysis of Cytoplasmic Domain of Podocyte Protein Neph1 Using Small/Wide Angle X-ray Scattering (SWAXS)

Mallik, Leena; Arif, Ehtesham; Sharma, Pankaj; Rathore, Yogendra Singh; Wong, Hetty N.; Holzman, Lawrence B.; Ashish, .; Nihalani, Deepak

doi:10.1074/jbc.m111.284927

Cited by 13 publications

(32 citation statements)

References 25 publications

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“…Proteins, including glutathion S-transferase (GST)-Neph1 cytoplasmic domain (CD), His-Neph1-CD-wt, His-Neph1-CD-R750E, His-Neph1-CD-(Ϫ)PDZ, His-Neph1-CD-K761A, and His-Neph1-CD-Y762A, were expressed and purified from Escherichia coli BL21 cells (Stratagene). Details of the expression and purification protocol have been described previously (39). Expression and purification of His-ZO1-PDZ1 protein have also been described previously (14,39).…”

Section: Methodsmentioning

confidence: 99%

“…The SAXS intensity profiles were collected at the X9 beam line (National Synchrotron Light Source, Brookhaven National Laboratory, NY). Images were acquired on Pilatus detectors and processed as described earlier (39). For each experiment, 120 l of protein solutions (unliganded Myo1c, its fragments, Neph1-CD, and their mixtures) and their matched buffers were exposed to X rays in a quartz capillary flow cell.…”

Section: Methodsmentioning

confidence: 99%

“…The open-source product Pymol version 1.1 was used for graphical analysis and figure generation. The structure of His-Neph1-CD (39) was employed for docking on the composite model of Myo1c (39). Docking of His-Neph1-CD on Myo1c was done using the ZDOCK server, and the model of the 1:1 complex was considered based on agreement with the shape profile of the SAXS data-based envelope shape of the complex (46).…”

Section: Methodsmentioning

confidence: 99%

“…Details of the expression and purification protocol have been described previously (39). Expression and purification of His-ZO1-PDZ1 protein have also been described previously (14,39). Recombinant proteins Flag-Myo1c-FL and GFP-HisMyo1c-tail were produced in a baculovirus expression system as described previously (40,41).…”

Section: Methodsmentioning

confidence: 99%

“…The recombinant proteins-His-Neph1-CD, the Neph1-CD mutants (including His-Neph1-CD-R750A, His-Neph1-CD-K761A, HisNeph1-CD-Y762A, and His-Neph1-CD-PDZ), and His-ZO1-PDZ1-were expressed and purified in Escherichia coli BL21 cells (Stratagene) as described previously (39). Recombinant proteins Flag-Myo1c-FL and GFPHis-Myo1c-tail were separately mixed with 5 g of each Neph1 fusion protein, and the His-Neph1-CD/Myo1c complexes were pulled down using Neph1 antibody bound to protein G-agarose.…”

Section: Methodsmentioning

confidence: 99%

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Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1

Arif

Sharma

Solanki

et al. 2016

Molecular and Cellular Biology

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The Myo1c motor functions as a cargo transporter supporting various cellular events, including vesicular trafficking, cell migration, and stereociliary movements of hair cells. Although its partial crystal structures were recently described, the structural details of its interaction with cargo proteins remain unknown. This study presents the first structural demonstration of a cargo protein, Neph1, attached to Myo1c, providing novel insights into the role of Myo1c

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1

Arif

Sharma

Solanki

et al. 2016

Molecular and Cellular Biology

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Insights into open/closed conformations of the catalytically active human guanylate kinase as investigated by small-angle X-ray scattering

et al. 2015

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Bio-catalysis is the outcome of a subtle interplay between internal motions in enzymes and chemical kinetics. Small-angle X-ray scattering (SAXS) investigation of an enzyme’s internal motions during catalysis offers an integral view of the protein’s structural plasticity, dynamics, and function, which is useful for understanding allosteric effects and developing novel medicines. Guanylate kinase (GMPK) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms. It is also required for the intracellular activation of numerous antiviral and anticancer purine nucleoside analog prodrugs. Catalytically active recombinant human GMPK (hGMPK) was purified for the first time and changes in the size and shape of open/closed hGMPK were tracked by SAXS. The binding of substrates (GMP + AMPPNP or Ap5G or GMP + ADP) resulted in the compaction of size and shape of hGMPK. The structural changes between open and completely closed hGMPK conformation were confirmed by observing differences in the hGMPK secondary structures with circular dichroism spectroscopy.Graphical abstractElectronic supplementary materialThe online version of this article (doi:10.1007/s00249-015-1079-9) contains supplementary material, which is available to authorized users.

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Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

Solanki

Arif

Srivastava

et al. 2021

Journal of Biological Chemistry

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Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.

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Solution Structure Analysis of Cytoplasmic Domain of Podocyte Protein Neph1 Using Small/Wide Angle X-ray Scattering (SWAXS)

Cited by 13 publications

References 25 publications

Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1

Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1

Insights into open/closed conformations of the catalytically active human guanylate kinase as investigated by small-angle X-ray scattering

Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

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