Abstract:Highlights d Structural and functional studies of CNNM, a putative Mg 2+ transporter in human d Crystal structures of cytosolic fragments in open and closed conformations d Tight correlation between Mg 2+ -ATP binding and CBS-pair domain dimerization d Mg 2+ efflux is regulated by conformational changes associated with Mg 2+ -ATP binding
“…Only ADP exhibited a very weak effect on the FRET intensity, altering the intensity by 9.6%. Our result indicates that the interaction between CNNM3 and PRL2 is not regulated by the Mg-ATP complex, whereas a recent study indicated that the Mg-ATP complex is sensed by CNNM 8 . Figure 5 Effect of divalent cations and nucleotides on the FRET intensities.…”
The cyclin M (CNNM) family of Mg
2+
transporters is reported to promote tumour progression by binding to phosphatase of regenerating liver (PRL) proteins. Here, we established an assay for detection of the binding between the cystathionine-beta-synthase (CBS) domain of human CNNM3 (a region responsible for PRL binding) and human PRL2 using fluorescence resonance energy transfer (FRET) techniques. By fusing YPet to the C-terminus of the CNNM3 CBS domain and CyPet to the N-terminus of PRL2, we performed a FRET-based binding assay with purified proteins in multiwell plates and successfully detected the changes in fluorescence intensity derived from FRET with a reasonable
K
d
. We then confirmed that the addition of non-YPet-tagged CNNM3 and non-CyPet-tagged PRL proteins inhibited the changes in FRET intensity, whereas non-YPet-tagged CNNM3 with a mutation at the PRL2-binding site did not exhibit such inhibition. Furthermore, newly synthesized peptides derived from the CNNM loop region, with the PRL-binding sequences of the CNNM3 CBS domain, inhibited the interactions between CNNM3 and PRL2. Overall, these results showed that this method can be used for screening to identify inhibitors of CNNM-PRL interactions, potentially for novel anticancer therapy.
“…Only ADP exhibited a very weak effect on the FRET intensity, altering the intensity by 9.6%. Our result indicates that the interaction between CNNM3 and PRL2 is not regulated by the Mg-ATP complex, whereas a recent study indicated that the Mg-ATP complex is sensed by CNNM 8 . Figure 5 Effect of divalent cations and nucleotides on the FRET intensities.…”
The cyclin M (CNNM) family of Mg
2+
transporters is reported to promote tumour progression by binding to phosphatase of regenerating liver (PRL) proteins. Here, we established an assay for detection of the binding between the cystathionine-beta-synthase (CBS) domain of human CNNM3 (a region responsible for PRL binding) and human PRL2 using fluorescence resonance energy transfer (FRET) techniques. By fusing YPet to the C-terminus of the CNNM3 CBS domain and CyPet to the N-terminus of PRL2, we performed a FRET-based binding assay with purified proteins in multiwell plates and successfully detected the changes in fluorescence intensity derived from FRET with a reasonable
K
d
. We then confirmed that the addition of non-YPet-tagged CNNM3 and non-CyPet-tagged PRL proteins inhibited the changes in FRET intensity, whereas non-YPet-tagged CNNM3 with a mutation at the PRL2-binding site did not exhibit such inhibition. Furthermore, newly synthesized peptides derived from the CNNM loop region, with the PRL-binding sequences of the CNNM3 CBS domain, inhibited the interactions between CNNM3 and PRL2. Overall, these results showed that this method can be used for screening to identify inhibitors of CNNM-PRL interactions, potentially for novel anticancer therapy.
“…The Cystathionine-β-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) have been described to be involved in maintaining Mg2+ homeostasis. This family includes four proteins (CNNM1, CNNM2, CNNM3 and CNNM4) [6]. Among these, the CNNM2 protein, that was described in 2003 [7], is ubiquitously expressed, although mostly at the basolateral membrane in both the thick ascending limb of the Henle's loop, and the distal collecting tubule within the kidney where it is involved in Mg2+ reabsorption [8].…”
Section: Introductionmentioning
confidence: 99%
“…The exact mechanism by which these proteins facilitate the passing of Mg2+ through the cell membranes has been widely discussed [10,11]. These proteins could be themselves Mg2+ transporters [6,10] or regulate other proteins that transport Mg2+ [11].…”
Section: Introductionmentioning
confidence: 99%
“…Dimerization depends on Mg2+-ATP binding and is enhanced by phosphatases of regenerating liver (PRL) binding [13]. PRL binding is mediated by a loop from CBS-pair domain that extends into the phosphatase active site [6]. The CNBH domain does not bind cyclic nucleotides.…”
Section: Introductionmentioning
confidence: 99%
“…CNBH mediates CBS-par domains dimerization and is required for the Mg2+ efflux activity [ 14 ]. It has been proposed that the CNNM protein activity is regulated by conformational changes in the CBS-pair domain associated with Mg2+-ATP binding [ 6 ].…”
The maintenance of magnesium (Mg2+) homeostasis is essential for human life. The Cystathionine-β-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) have been described to be involved in maintaining Mg2+ homeostasis. Among these CNNMs, CNNM2 is expressed in the basolateral membrane of the kidney tubules where it is involved in Mg2+ reabsorption. A total of four patients, two of them with a suspected disorder of calcium metabolism, and two patients with a clinical diagnosis of primary tubulopathy were screened for mutations by Next-Generation Sequencing (NGS). We found one novel likely pathogenic variant in the heterozygous state (c.2384C>A; p.(Ser795*)) in the CNNM2 gene in a family with a suspected disorder of calcium metabolism. In this family, hypomagnesemia was indirectly discovered. Moreover, we observed three novel variants of uncertain significance in heterozygous state in the other three patients (c.557G>C; p. (Ser186Thr), c.778A>T; p.(Ile260Phe), and c.1003G>A; p.(Asp335Asn)). Our study shows the utility of Next-Generation Sequencing in unravelling the genetic origin of rare diseases. In clinical practice, serum Mg2+ should be determined in calcium and PTH-related disorders.
Background: Hypomagnesemia, seizures, and mental retardation (HSMR) syndrome is a rare genetic disease. Presently, only 24 cases have been reported and the clinical features of the disease are yet to be fully described, thereby making diagnosis challenging.Methods: Trio-whole-exome sequencing was used for the patient and her parents, and the structure of the variant protein was analyzed by molecular dynamics. Finally, the characteristics of HSMR were summarized by reviewing the previous literature.
Results:The main disease manifestations in the patient were seizures, liver function damage, hypomagnesemia, atrial septal defect, and sinus arrhythmia.A novel mutation in CNNM2 (c.566A>G/p.Tyr189Cys) was identified by genetic detection. The parents were wild type, and the mutation was rated as pathogenic by American College of Medical Genetics and Genomics guidelines. Ab initio modeling and molecular dynamics simulation show that the mutation destroys the surrounding hydrogen bonds, which may reduce the local stability of the protein structure. In the previous literature, only 24 children with HSMR have been reported, mainly manifested as hypomagnesemia, mental retardation, seizures, and language and motor impairment.
Conclusion:We have reported the second case of HSMR in the Chinese population, which further expands the phenotypic spectrum of congenital heart disease and the variation spectrum of CNNM2.
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