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Direct fusion of subunits of heterodimeric nitric oxide sensitive guanylyl cyclase leads to functional enzymes with preserved biochemical properties: evidence for isoform specific activation by ciguates.

Abstract：

:Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of an α and a β subunit. Two heterodimeric enzymes are known to be important for NO-signalling in humans: α(1)/β(1) and α(2)/β(1). No difference had so far been detected with respect to their pharmacological properties, but as we show in the present paper the new drugs cinaciguat and ataciguat activate the α(1)/β(1) form more effectively. Recent evidence suggests that homodimeric complexes of α and β subunits exist in vivo and that these non-heterodimerizing subunits have a separate function from cGMP signaling. To isolate the effect of the α(1)/β(1) or α(2)/β(1) heterodimeric enzyme in overexpression experiments from potential effects of non-heterodimerizing α(1), β(1) or α(2) subunits, we cloned constructs that guarantee a 1:1 stochiometry between α and β subunits and rule out the presence of homodimers. The carboxy-terminus of the β(1) subunit was directly fused to the amino-terminus of either the α(1) or α(2) subunit. The two different "conjoined" NOsGCs faithfully reproduced the biochemical and pharmacological properties of the α(1)/β(1) and α(2)/β(1) heterodimeric enzymes including the differential activation by ciguat-activators. Conjoined NOsGCs can be used for isoform specific overexpression in transgenic animals and therapeutic overexpression may be an application in the future. In both cases possible side effects of homodimeric α or β subunits are avoided. Crystallization with the goal of structure determination may also be easier for conjoined NOsGCs because enzyme preparations are more homogenous and are free of "contaminating" homodimers.

journal_name

Biochem Pharmacol

journal_title

Biochemical pharmacology

authors

Haase N,Haase T,Kraehling JR,Behrends S

doi

10.1016/j.bcp.2010.08.007

subject

Has Abstract

pub_date

2010-12-01 00:00:00

pages

1676-83

issue

eissn

0006-2952

issn

1873-2968

pii

S0006-2952(10)00616-7

journal_volume

pub_type

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Direct fusion of subunits of heterodimeric nitric oxide sensitive guanylyl cyclase leads to functional enzymes with preserved biochemical properties: evidence for isoform specific activation by ciguates.