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9 results on '"Sidhu, Ateesh"'

1. Structural studies on protein disulphide isomerase

Author

Sidhu, Ateesh

Subjects
571.4, QP Physiology
Abstract

Protein disulphide isomerase (POI; EC 5.3.4.1) is a multifunctional enzyme which resides in the lumen of the endoplasmic reticulum (ER). It is approximated that over one-third of all human proteins fold in the ER. POI is one of the main folding catalysts, specifically facilitating native disulphide bond formation. POI has four domains, three of which have been solved and all possessing a thioredoxin-type fold consisting of the J3-a-J3-a-J3-a-J3-J3-a structure. Due to the intransigent nature of the b' domain of POI, it remains structurally unelucidated. The b' domain is vitally important as it holds the principal binding site and is essential for POI activity. The investigation of the two species which were produced during expression of most b' domain containing constructs led to the further biophysical analysis and identification of the monomer and dimer species. It was found that fractionation of the monomer and dimer species was vital in obtaining high resolution NMR data. An elaborate method using assignments from the b domain and b'x were used, and proved very effective in achieving the goal of full backbone assignment of the large 27.5 kOa bb'x protein molecule. The fruit of this labour is that it allowed further probing dynamic and more detailed molecular NMR analysis. NMR analysis identified evidence that the bb'x monomer species can exist in two forms, the closed form where the x-region binds to the b' domain and open form where the x-region is unbound. Chemical shift analysis revealed several key residues involved in protein flexibility and chemical shift mapping revealed the interface between the b domain and b'x. Defining the interface described here offers a method to model the full length POI domain structural organisation; which can be used to more clearly define POI function and how the domains of POI are coordinated in protein function. More detailed NMR analysis of relaxation dynamics (T1 and T2) revealed significantly differing motions attributed to the b, b' domains and x-region in the bb'x construct. The slower b' domain motion is believed to be related to the substrate binding function, where the b' domain appears to be gently flexing, in search for a substrate molecule.

Published
2008

3. Autoregulation of Parkin activity through its ubiquitin-like domain

Author

Chaugule, Viduth K, Burchell, Lynn, Barber, Kathryn R, Sidhu, Ateesh, Leslie, Simon J, Shaw, Gary S, and Walden, Helen

Subjects
Have You Seen...?, Sequence Homology, Amino Acid, Protein Conformation, Ubiquitin, Ubiquitin-Protein Ligases, Molecular Sequence Data, Parkinson Disease, Article, Gene Expression Regulation, Enzymologic, Peptide Fragments, nervous system diseases, Mutation, Homeostasis, Humans, Amino Acid Sequence
Abstract

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING-InBetweenRING-RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity.

Published
2011

8. Alternative Conformations of the x Region of Human Protein Disulphide-Isomerase Modulate Exposure of the Substrate Binding b’ Domain

Author

Nguyen, Van Dat, primary, Wallis, Katrine, additional, Howard, Mark J., additional, Haapalainen, Antti M., additional, Salo, Kirsi E.H., additional, Saaranen, Mirva J., additional, Sidhu, Ateesh, additional, Wierenga, Rik K., additional, Freedman, Robert B., additional, Ruddock, Lloyd W., additional, and Williamson, Richard A., additional

Published
2008
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9. Reconstitution of Human Ero1-Lα/Protein-Disulfide Isomerase Oxidative Folding Pathway in Vitro POSITION-DEPENDENT DIFFERENCES IN ROLE BETWEEN THE a AND a' DOMAINS OF PROTEIN-DISULFIDE ISOMERASE.

Author

Lei Wang, Sheng-jian Li, Sidhu, Ateesh, Li Zhu, Yi Liang, Freedman, Robert B., and Chih-chen Wang

Subjects
*ISOMERASES, *ENDOPLASMIC reticulum, *THIOREDOXIN, *OXIDASES, *HYDROGEN peroxide, *MUTAGENESIS, *CALORIMETRY
Abstract

Protein-disulfide isomerase (PDI), a critical enzyme responsible for oxidative protein folding in the eukaryotic endoplasmic reticulum, is composed of four thioredoxin domains a, b, b', a', and a linker x between b' and a'. Ero1-Lα, an oxidase for human PDI (hPDI), has been determined to have one molecular flavin adenine dinucleotide (FAD) as its prosthetic group. Oxygen consumption assays with purified recombinant Ero1-Lα revealed that it utilizes oxygen as a terminal electron acceptor producing one disulfide bond and one molecule of hydrogen peroxide per dioxygen molecule consumed. Exogenous FAD is not required for recombinant Ero1-Lα activity. By monitoring the reactivation of denatured and reduced RNase A, we reconstituted the Ero1-Lα/hPDI oxidative folding system in vitro and determined the enzymatic activities of hPDI in this system. Mutagenesis studies suggested that the a' domain of hPDI is much more active than the a domain in Ero1-Lα-mediated oxidative folding. A domain swapping study revealed that one catalytic thioredoxin domain to the C-terminal of bb'x, whether a or a', is essential in Ero1-Lα-mediated oxidative folding. These data, combined with a pull-down assay and isothermal titration calorimetry measurements, enabled the minimal element for binding with Ero1-Lα to be mapped to the b'xa' fragment of hPDI. [ABSTRACT FROM AUTHOR]

Published
2009
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