The document discusses the Na+/K+-ATPase pump, which imports 2K+ and exports 3Na+ across cell membranes using ATP hydrolysis, and the role of FXYD proteins in modulating the kinetic properties of the pump. It outlines a proposed study to express and purify human Na+/K+ pump isoforms and FXYD proteins, examine the effects of FXYD proteins on pump kinetics, and screen natural compounds for isoform-selective inhibitors/activators of the human pump. Materials, methods, and references are provided to support the proposed work.
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My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
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A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
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3. Na+/K+-ATPase is an integral membrane protein.
It imports 2K+ and export 3Na+ for the cell at the expanse of hydrolysis of ATP, and
establish electrochemical gradient across the cell membrane.
The pump also plays a crucial role in some specialized functions such as: Renal sodium
reabsorption, muscle contraction and neuronal excitability.
In cells pump exists as heterotrimer consists of one catalytic a subunit, one chaperone
like b-subunit and regulatory FXYD protein also known as (g subunit).
The pump potentially found in 96 isoforms (4 a-subunit and 3 b-subunits and 7 FXYDs)
with different transport properties depends upon the tissue’s need.
Uses ~30% and ~70% of total ATP consumption of the muscles and nerve cells
respectively.
Na+/ K+-ATPase
4. FXYD
FXYD proteins are a family of seven small single transmembrane proteins (FXYD1-7).
Because of presence of conserved FXYD sequence in all homologues, These small single
transmembrane domain proteins are known as FXYD proteins.
FXYD proteins play role in proper handling of electrochemical gradient by the transport of
Na+ and K+ with Na+/K+-ATPase.
FXYD proteins modulate the kinetic properties of Na+/K+-ATPase and thus adapting the Na+,
K+ transport against the gradient according to the requirement of specific tissue.
The effects of FXYD proteins on parameters such as K1/2 Na+, K1/2K+, KmATP and Vmax are
modest (2-3fold).
6. The Na+/K+-ATPase reaction cycle
Mishra, N.K., et al., J Biol Chem. 2015 Nov 27;290(48):28746-59 ;
Mishra, N.K., et al., J Biol Chem. 2011 Mar 18;286(11):9699-712
Cirri, E. Et al., Biochemistry. 2011 May 10;50(18):3736-48.
7. Structure of the Shark rectal gland Na+/K+-ATPase
Shindoa et al., 2009 Nature
8. Study Proposed
• Establishing expression and purification platform for all human Na+/K+-ATPase
isoforms.
• Expression and purification of human FXYD proteins
• Effect of FXYD proteins upon kinetic properties of Na+/K+-ATPase.
• Research’s main focus will be on α2β1, α3β1 and α4β1 isozymes.
• Search for isoform-selective inhibitor/activator of human Na+/K+-ATPase by
natural compound library screening
9. Material will be needed
Cells:
• E. coli for the subcloning purpose (DH5a, XL1blue)
• E. coli for protein expression (C41, C43, Rosetta, Bl21)
• Pichia pastoris (Protein expression)
Reagents:
• Buffers and other common reagents.
• Molecular cloning reagents.
• Media components.
• Protein purification reagents (Affinity resins, columns)
• Enzyme assay reagents.
10. Strugatsky D et al. J. Biol. Chem. 2003;278:46064-46073; Mishra, N.K., et al., 2015 Nov 27;290(48):28746-59 ; Mishra, N.K.,
et alJ Biol Chem. 2011 Mar 18;286(11):9699-712
11. hFXYD1, FXYD2b and FXYD4 were cloned
into pET28-TEVH
(a modification of pET28)
6x His TEV site Gene (PLM, CHIF, g-a)
Expression was done in E. coli - BL21(DE3), C41 and C43.
Mishra, N.K., et al., J Biol Chem. 2015 Nov 27;290(48):28746-59 ;
Mishra, N.K., et al., J Biol Chem. 2011 Mar 18;286(11):9699-712
12. FXYDs expression
• FXYD1,2 and 4 clones in the
pET28a vector will be provided by
Prof. Karlish’s lab.
• FXYD3,5,6 and 7 genes will be
synthesized chemically.
• Chemically synthesized genes will
be inserted (cloned) between
appropriate cloning sites (Kpn1 and
NotI for FXYD1,2 and 4)
13. Methods:
• I will get α1β1, α2β1, and α3β1 isozyme plasmids from the Weizmann Institute of
Science.
• The gene of α4 will be synthesized chemically followed by its insertion in the plasmid
of α1β1 by replacing the α1 gene with α4.
• FXYD1,2 and 4 plasmids will be provided by Prof. Karlish’s laboratory and the rest of
the FXYD (3,5,6 and 7) genes will also be synthesized chemically and expressed in
E.coli.
• Complete isozyme will be reconstituted by mixing purified αβ and FXYD proteins.
• ATPase assays will be performed by the malachite green method.
• To study the kinetics of conformational changes selective isoforms will be labeled by
the fluorescent probe (FITC).
• Enzyme functional kinetics will be performed by ATPase assay.
14. Mishra, N.K., et al., J Biol Chem. 2015 Nov 27;290(48):28746-59 ;
Mishra, N.K., et al., J Biol Chem. 2011 Mar 18;286(11):9699-712
Editor's Notes
Expression vector pHIL-D2(α/β). Note NotI sites used to linearize the expression cassette prior to transformation of P. pastoris. bla, β-lactamase; kan, kanamycin resistance gene; His4, histidinol dehydrogenase.
